small gtpases and their regulators, part f

Preface As with the Rho and Rab branches of the Ras superfamily of small GTPases, research interest in the Ras branch has continued to expand dramatically into new areas and to embrace new themes since the last Methods in Enzymology Volume 255 on Ras GTPases was published in 1995. First, the Ras branch has expanded beyond the original Ras, Rap, and Ral members. New members include M-Ras, Rheb, Rin, and Rit. Second, the signaling activities of Ras are much more diverse and complex than appreciated previously. In particular, while the Raf/MEK/ERK kinase cascade remains a key signaling pathway activated by Ras, it is now appreci- ated that an increasing number of non-Raf effectors also mediate Ras family protein function. Third, it is increasingly clear that the cellular functions regulated by Ras go beyond regulation of cell proliferation, and involve regulation of senescence and cell survival and induction of tumor cell invasion, metastasis, and angiogenesis. Fourth, another theme that has emerged is regulatory cross talk among Ras family proteins, including both GTPase signaling cascades that link signaling from one family member to another, as well as the use of shared regulators and effectors by different family members. Concurrent with the expanded complexity of Ras family biology, bio- chemistry, and signaling have been the development and application of a wider array of methodology to study Ras family function. While some are simply improved methods to study old questions, many others involve novel approaches to study aspects of Ras family protein function not studied previously. In particular, the emerging application of techniques to study Ras regulation of gene and protein expression represents an important direction for current and future studies. Consequently, Methods in Enzy- mology, Volumes 332 and 333 cover many of the new techniques that have emerged during the past five years. We are grateful for the efforts of all our colleagues who contributed to these volumes. We are indebted to them for sharing their expertise and experiences, as well as their time, in compiling this comprehensive series of chapters. In particular, we hope these volumes will provide valuable references and sources of information that will facilitate the efforts of newly incoming researchers to the study of the Ras family of small GTPases. CHANNING J. DER ALAN HALL WILLIAM E. BALCH xiii Contributors to Volume 332 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. NATALIE G. AHN (31), Department of Chem- istry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309 GORDON ALTON (23), Celgene Corporation Signal Research Division, Department of Imformatics and Functional Genomics, San Diego, California 92121 DOUGLAS A. ANDRES (14, 15), Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084 M. JANE ARBOLEDA (27), Onyx Pharmaceuti- cals, Richmond, California 94806 AMI ARONHEIM (20), Department of Molecu- lar Genetics, The B. Rappaport Faculty of Medicine, Israel Institute of Technology, Haifa 31096, Israel BRYDON L. BENNEqT (32), Signal Pharmaceu- ticals, Inc., San Diego, California 92121 W. ROBERT BISHOP (8), Department of Tumor Biology, Schering Plough Research Insti- tute, Kenilworth, New Jersey 07033 BENJAMIN BOETTNER (11), Cold Spring Har- bor Laboratory, Cold Spring Harbor, New York 11724 GIDEON BOLLAG (7, 19), Onyx Pharmaceuti- cals, Richmond, California 94806 MICHELLE A. BOODEN (4), Lineberger Com- prehensive Cancer Center, CB-7295, Uni- versity of North Carolina, Chapel Hill, North Carolina 27599 JANICE E. Buss (4), Department of Biochemis- try, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 ANDREW D. CATLING (28), Department of Mi- crobiology and Cancer Center, University of Virginia Health Sciences Center, Char- lottesville, Virginia 22908-0734 MEENA A. CHELLAIAH (2), Renal Division, Barnes-Jewish Hospital, Washington Uni- versity School of Medicine, St. Louis, Mis- souri 63110 JONATHAN CHERNOFF (22), Division of Basic Science, Fox Chase Cancer Center, Phila- delphia, Pennsylvania 19111 YONO-JIG CHO (18), Vanderbilt-Ingram Can- cer Center, Nashville, Tennessee 37232-6838 YUN-JUNG CHOI (7, 19), Onyx Pharmaceuti- cals, Richmond, California 94806 EDWIN CHOY (3), Departments of Medicine and Cell Biology, New York University School of Medicine, New York, New York 10016 MELANIE H. COBB (29), Department of Phar- macology, University of Texas Southwest- ern Medical Center, Dallas, Texas 75235- 9041 JOHN COLICELLI (10), Department of Biologi- cal Chemistry and Molecular Biology Insti- tute, UCLA School of Medicine, Los Angeles, California 90095 ADRIENNE D. Cox (1, 23), Department of Radiation Oncology and Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 ROGER J. DAVIS (24), Howard Hughes Medi- cal Institute, Department of Biochemistry and Molecular Biology, University of Mas- sachusetts Medical School, Program in Mo- lecular Medicine, Worcester, Massachu- setts 01605 CHANNING J. DER (1, 17), Lineberger Com- prehensive Cancer Center, Department of Pharmacology, University of North Caro- lina, Chapel Hill, North Carolina 27599 STEVEN F. DOWDY (2), Departments of Pa- thology and Medicine, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110 ix X CONTRIBUTORS TO VOLUME 332 DEREK EBERWEIN (27), Bayer Corporation, West Haven, Connecticut 06516-4175 SCOTT T. EaLEN (28), Department of Microbi- ology and Cancer Center, University of Virginia Health Sciences Center, Charlottes- ville, Virginia 22908-0734 JAMES J. FIORDALISI (1), Departments of Ra- diation, Oncology, and Pharmacology, Uni- versity of North Carolina, Chapel Hill, North Carolina 27599 DANIEL G. GIOELI (26), Department of Micro- biology and Cancer Center, University of Virginia Health Sciences Center, Charlottes- ville, Virginia 22908 ERICA A. GOLEMIS (5, 22), Division of Basic Science, Fox Chase Cancer Center, Phila- delphia, Pennsylvania 19111 SAID A. GOUELI (25), Signal Transduction Group, Research and Development Depart- ment, Promega Corporation, Madison, Wis- consin 53711, and Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine, Madison, Wisconsin 53711 GASTON G. HABETS (19), Onyx Pharmaceuti- cals, Richmond, California 94806 CHRISTIAN HERRMANN (11), Max Planck In- stitute for Molecular Physiology, 44227 Dortmund, Germany BARBARA HIBNER (27), Bayer Corporation, West Haven, Connecticut 06516-4175 KEITH A. HRUSKA (2), Renal Division, Barnes-Jewish Hospital, Washington Uni- versity School of Medicine, St. Louis, Mis- souri 63110 BRUCE W. JARVIS (25), Signal Transduction Group, Research and Development Depart- ment, Promega Corporation, Madison, Wis- consin 53711 HAKRYUL Jo (18), Vanderbilt-lngram Cancer Center, Nashville, Tennessee 37232-6838 RONALD L. JOHNSON II (1), Departments of Radiation, Oncology, and Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 KIRAN J. KAUR (21), Department of Cell Biol- ogy, University of Texas Southwestern Med- ical Center, Dallas, Texas 75390 BRIAN K. KAY (6), Department of Pharmacol- ogy, University of Wisconsin, Madison, Wisconsin 53706-1532 AKIRA KIKUCHI (9), Department of Biochem- istry, Hiroshima University School of Medi- cine, Hiroshima 734-8551, Japan PAUL T. KIRSCHMEIER (8), Department of Tu- mor Biology, Schering Plough Research In- stitute, Kenilworth, New Jersey 07033 MARC KNEPPER (19), Advanced Medicine, Inc., San Francisco, California 94080 SHINYA KOYAMA (9), Department of Bio- chemistry, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan PENG LIANG (18), Vanderbilt-lngram Cancer Center, Nashville, Tennessee 37232-6838 DAN LIU (13), Verna and Marts McLean De- partment of Biochemistry and Molecular Biology, Baylor College of Medicine, Hous- ton, Texas 77030 MARK LYNCH (7), Bayer Research Center, West Haven, Connecticut 06516 JOHN F. LYONS (27), Onyx Pharmaceuticals, Richmond, California 94806 GWENDOLYN M. MAHON (16), Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103-2714 MARTIN MCMAHON (30), Cancer Research Institute and Department of Cellular and Molecular Pharmacology, University of California San Francisco/Mt. Zion Com- prehensive Cancer Center, San Francisco, California 94115 OLGA V. MITINA (22), Department of Molecu- lar Biology and Medical Biotechnology, Russian State Medical University, Mos- cow, Russia BRION W. MURRAY (32), Agouron Pharma- ceuticals, San Diego, California 92121-1408 THERESA STINES NAHREINI (31), Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Col- orado, Boulder, Colorado 80309 CONTRIBUTORS TO VOLUME 332 xi MICHAEL NIEDBALA (7), Bayer Research Cen- ter, West Haven, Connecticut 06516 ANNE K. NORTH (7), Onyx Pharmaceuticals, Richmond, California 94806 JIN-KEON PAl (8), Department of Tumor Biol- ogy, Schering Plough Research Institute, Kenilworth, New Jersey 07033 MARK PHILIPS (3), Departments of Medicine and Cell Biology, New York University School of Medicine, New York, New York 10016 SCOTT POWERS (17), Tularik Genomics, Greenlawn, New York 11740 KATHERYN A. RESING (31), Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309 DENNIS Z. SASAKI (32), Signal Pharmaceuti- cals, Inc., San Diego, California 92121 TAKEHIKO SASAZUKI (19), Medical Institute of Bioregulation, Kyushu University, Fuku- oka 812, Japan HANS J. SCHAEFFER (28), MDC, Gruppe W. Birchmeier, 13125 Berlin, Germany ILYA G. SEREBRIISKII (22), Division of Basic Science, Fox Chase Cancer Center, Phila- delphia, Pennsylvania 19111 JANIEL M. SHIELDS (17), Department of Phar- macology, Lineberger Comprehensive Can- cer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295 SENJI SHIRASAWA (19), Medical Institute of Bioregulation, Kyushu University, Fukuoka 812, Japan ZHOU SONGYANG (12, 13), Verna and Marts McLean Department of Biochemistry and Molecular Biology, Baylor College of Medi- cine, Houston, Texas 77030 JOHN T. STICKNEY (4), Department of Cell Biology, Neurobiology, and Anatomy, Uni- versity of Cincinnati Medical Center, Cin- cinnati, Ohio 45267-0521 JAINA SUMORTIN (19), Onyx Pharmaceuticals, Richmond, California 94806 MARC SYMONS (7), The Picower Institute for Medical Research, Manhasset, New York 11030 GARABET G. TOBY (5), Division of Basic Sci- ence, Fox Chase Cancer Center, Philadel- phia, Pennsylvania 19111, and Cell and Mo- lecular Biology Group, University of Pennsylvania School of Medicine, Philadel- phia, Pennsylvania 19104-6064 NICHOLAS S. TOLWlNSKI (31), The Graduate College, Princeton University, Princeton, New Jersey 08544 L. GERARD TOUSSAINT III (23), Distinguished Medical Scholar Program, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 AYLIN S. LILK0 (1), Department of Pharma- cology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 LINDA VAN AELST (11), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 ADAMINA VOCERO-AKBANI (2), Departments of Pathology and Medicine, Howard Hughes Medical Institute, Washington Uni- versity School of Medicine, St. Louis, Mis- souri 63110 YING WANG (10), Department of Biological Chemistry and Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California 90095 MICHAEL J. WEBER (26, 28), Department of Microbiology and Cancer Center, Univer- sity of Virginia Health Sciences Center, Charlottesville, Virginia 22908-0734 JOHN K. WESTWICK (23), Celgene Corporation Signal Research Division, Department of Cell Signaling, San Diego, California 92121 MICHAEL A. WHITE (21), Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 IAN P. WHITEHEAD (16), Department of Mi- crobiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103-2714 ALAN J. WHITMARSH (24), Howard Hughes Medical Institute, Department of Biochem- istry and Molecular Biology, University of Massachusetts Medical School, Program in xii CONTRIBUTORS TO VOLUME 332 Molecular Medicine, Worcester, Massachu- setts 01605 DAVID WHYTE (8), Sugen Inc., South San Francisco, California 94080 Jueiz L. WmSBACHER (29), Department of Pharmacology, University of Texas South- western Medical Center, Dallas, Texas 75235-9041 OSWALD WILSON (8), Department of Tumor Biology, Schering Plough Research Insti- tute, Kenilworth, New Jersey 07033 MONTAROP YAMABHAI (6), School of Bio- technology, Suranaree University of Tech- nology, Institute of Agricultural Technol- ogy, Nakhon Ratchasima 30000, Thailand MAJA ZECEVIC (26), Department of Microbi- ology and Cancer Center, University of Vir- ginia Health Sciences Center, Charlottes- ville, Virginia 22908 HONG ZHANG (18), Vanderbilt-Ingram Can- cer Center, Nashville, Tennessee37232-6838 [ 1] MAMMALIAN EXPRESSION VECTORS FOR Ras 3 [i] Mammalian Expression Vectors for Ras Family Proteins: Generation and Use of Expression Constructs to Analyze Ras Family Function By JAMES J. FIORDALISI, RONALD L. JOHNSON II, AYLIN S. I, JLKO, CHANNING J. DER, and ADRIENNE D. Cox Introduction Cell-based assays are useful for the characterization of Ras family struc- ture-function relationships, identification of upstream regulators and down- stream effectors, characterization of signaling inputs and outputs, analysis of the role of Ras family proteins in normal and aberrant cellular metabo- lism, and evaluation of potential anticancer agents. Common to all such studies is the need to express the protein(s) of interest within a cell. This is accomplished through the use of plasmid vectors into which are placed the coding sequences of the proteins to be studied, and which can then be introduced into cells by a variety of methods. Protein expression plasmid vectors contain signal sequences required for transcription and translation of the target protein (i.e., promoter elements, polyadenylation sites, etc.) as well as origins of replication for maintenance of the plasmid. Expression vectors have been developed with a variety of features, including selectable markers and sequences encoding epitope tags that are recognized by specific antibodies, which facilitate the subsequent analysis of protein expression and function. Not all vectors function equally well in different assay systems, even if the sequences being expressed are identical. Similarly, not all proteins are expressed equally well in the same vector. Moreover, the reasons for these differences are not well understood and can be determined only by trial and error. Therefore, choosing the optimum vector for a given protein and assay system can be an empirical and time-consuming endeavor. Undoubt- edly, such factors as the identity of the cell line, the gene of interest, the biological readout, as well as others all contribute to variability in the usefulness of the vector. In this chapter, we attempt to provide readers with a starting point from which to choose the most appropriate vector for their particular proteins of interest and intended uses. We present some observations concerning the strengths and weaknesses of several mammalian protein expression vectors, both commercially available and "homemade." Because there are many vectors currently in use, as well as new vectors and assay systems Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. METHODS IN ENZYMOLOGY, VOL. 332 0076-6879/00 $35.00 4 PROTEIN EXPRESSION AND PROTEIN-PROTEIN INTERACTIONS [ 1] continually being developed, it is not possible to present a comprehensive physical or functional evaluation of all vectors under all circumstances. In this work we identify and discuss most of the major factors that should be considered. In addition to discussing the advantages and disadvantages of particular features of mammalian protein expression vectors, we also compare and contrast them functionally with respect to biological readouts commonly used in the study of Ras protein function, including protein expression, signaling activity in enzyme-linked transcriptional trans-activa- tion reporter assays, and transforming ability in fibroblast focus-forming assays. In all cases we use activated, oncogenic Ras proteins as the model system. Because the choice of vector will be influenced by, among other things, the ease with which protein-coding sequences can be introduced into them, we also discuss several techniques for generating and manipulating protein expression constructs. Finally, we discuss several methods for intro- ducing plasmid DNA into mammalian cells, including transfection with a variety of reagents and infection using retroviral packaging vectors. Properties to Consider in Choosing a Vector Promoter In choosing a mammalian protein expression vector (Table Ii-8), the most important factor to consider is whether the plasmid will express the protein of interest to the desired level in the cell type to be used. Sometimes the highest possible protein expression levels are desired, usually in order to maximize the biological effect being studied. In other cases, lower levels are desired, usually either to achieve more physiologically relevant levels or to minimize toxicity. Protein expression is controlled primarily by the transcriptional promoter region of the vector, which contains elements necessary for transcription (such as binding sites for transcription factors that recruit RNA polymerase) and translation (especially the Kozak se- t M. A. White, C. Nicolette, A. Minden, A. Polverino, L. Van Aelst, M. Karin, and M. H. Wigler, Cell 80, 533 (1995). 2 R. R. Mattingly, A. Sorisky, M. R. Brann, and I. G. Macara, MoL Cell. Biol. 14, 7943 (1994). 3 j. p. Morgenstern and H. Land, Nucleic Acids Res. 18, 1068 (1990). 4 W. S. Pear, G. P. Nolan, M. L. Scott, and D. Baltimore, Proc. Natl. Acad. ScL U.S.A. 90, 8392 (1993). 5 I. Whitehead, H. Kirk, C. Tognon, G. Trigo-Gonzalez, and R. Kay, J. Biol. Chem. 270, 18388 (1995). 6 C. L. Cepko, B. Roberts, and R. C. Mulligan, Cell 37, 1053 (1984). 7 j. A. Southern, D. F. Young, F. Heaney, W. K. Baumgartner, and R. E. Randall, J. Gen. Virol. 72, 1551 (1991). 8 A. Yen, M. Williams, J. D. Platko, C. Der, and M. Hisaka, Eur. J. Cell Biol. 65, 103 (1994). [ 1] MAMMALIAN EXPRESSION VECTORS FOR Ras 5 quence 9) of the coding sequence. Most promoters found in expression vectors are derived from viral promoters that induce the high rates of protein expression necessary for viral replication. The cytomegalovirus (CMV) promoter, the mouse mammary tumor virus long terminal repeat promoter (MMTV LTR), and the Moloney murine leukemia virus promoter LTR (Mo-MuLV LTR) are commonly used viral promoters. The CMV promoter generally works well in cell lines derived from primate tissues such as human embryonic kidney cells (HEK293), human breast epithelial cells (T-47D, MCF-7, and MCF-10A), and monkey kidney cells (COS-7), but works less well in cells of rodent origin, such as mouse fibroblasts (NIH 3T3, Ratl, and Rat2) and rat intestinal epithelial cells (RIE-1). The reverse is true of the MMTV LTR and the Mo-MuLV LTR promoters. Naturally, there are always exceptions to such a rule; for exam- ple, we have found that pZIP-NeoSV(X)l-based constructs work well in T-47D cells but not in 293 or COS cells. Protein expression levels should always be confirmed directly for each expression construct in the cells of interest, using Western blot analysis or a similar method. Constitutive versus Inducible Protein Expression Although most vectors express proteins in a constitutive fashion, protein expression in some vectors is controlled by promoters that contain inducible elements that bind either repressor proteins or inducers that can be inacti- vated or induced, respectively, by exposure to exogenously added inducing agents. Until then, protein expression does not occur. We have more experi- ence with dexamethasone-inducible vectors 3 (Table I); other common in- ducible elements are responsive to tetracycline, 1°'11 isopropyl-/3-o-thiogalac- topyranoside (IPTG), 12 and ecdysone (see Ref. 13 and [19] in this volumel4). Inducible protein expression is desirable if the protein of interest is toxic or otherwise growth inhibitory to the cell, in which case, stable transfection of cells with a vector expressing this protein constitutively would be impossi- ble. Moreover, any transient or temporally distinct cellular phenotype caused by the expression of the protein can be evaluated better if protein expression can be turned on and off relatively rapidly. 9 M. Kozak, Nucleic Acids Res. 9, 5233 (1981). 10 L. Chin, A. Tam, J. Pomerantz, M. Wong, J. Holash, N. Bardeesy, Q. Shen, R. O'Hagan, J. Pantginis, H. Zhou, J. W. Horner II, C. Cordon-Cardo, G. D. Yancopoulos, and R. A. DePinho, Nature (London) 400, 468 (1999). 11 H. S. Liu, C. H. Lee, C. F. Lee, I. J. Su, and T. Y. Chang, BioTechniques 24, 624 (1998). 12 M. A. Wani, X. Xu, and P. J. Stambrook, Cancer Res. 54, 2504 (1994). 13 M. J. Calonge and J. Massague, J. BioL Chem. 274, 33637 (1999). 14 G. G. Habets, M. Knepper, J. Sumortin, Y J. Choi, T. Sasazuki, S. Shirasawa, and G. Bollag, Methods Enzymol. 332 [19] 2001 (this volume). 6 PROTEIN EXPRESSION AND PROTEIN-PROTEIN INTERACTIONS [1] ~2 Z 0 m ~.~ a rn M m e~ ~ ~ooo .~'~ ~~ m d < <<<<<<~<<<< m ~D O o/ O , a O ,o >. I O O ZZ t.) O O o E~ << [1] MAMMALIAN EXPRESSION VECTORS FOR Ras 7 O.Oc~ ~.~o .~ o ~,-, ~" c.,) "~ ~ ~~o" " ~- " ~. ~°° .,~>.~=~ ~ ,~ ~'~- • ¢.) 0 .,~ ~l c~ ¢) 0" ~ '~ ~.~ o ~o0 =l ,.~ ~ -"~"~ 0 ~ .0 ~ oj~ 0 ~ = ~.~ ~ ~ c~ ~ ~=~_ ~o ~.~ ~=°o-~ ~ ~ o = ~'~" ~ ~.~,-o ,~ ~ ~ ~ = ~ ~ ~ ~ , ~ 0 .~ I.a.~ ~ ,-~ 0 o ~= ~ ~ ~ ~.~=~ ~" ~ .~ v ~ ~ ~ £.) ~ 0 "~ "d 0 ~ ~I -~ ~ I ~ ~.~ ~ .~ .~ ~-~ ~-~ > .~: "~ c~ ~ = ~ o~ ~ ~-0.= ° ~-c ~ ~ ~ oo =.~ ~.~ x >~ >'~, ~ ~ ~o ~o ~ ~'0,~ ~ ~.~ ~'~ ~ o [...]... EXPRESSION AND P R O T E I N - P R O T E I N INTERACTIONS [ 1! more efficient extraction of DNA from the gel than does Tris-borate/ EDTA (TBE) After purification from the gel slice, 5% of each purified fragment is run on another agarose gel to confirm purity and estimate concentration Dephosphorylation of Digested Vector If two restriction enzymes are used to create different ends for directional cloning of... chloride transfection, suggesting that transient transfection assays can also benefit from their use In particular, consistent, high transfection efficiencies are required for reporter assays, and in cells such as RIE-1 this is not achievable in our hands without use of Superfect or FuGENE In contrast, NIH 3T3 fibroblasts transfect well with calcium and we do not use lipid reagents with these cells For experiments... amounts of DNA, the efficiency of the transfection method chosen is of prime importance The simplicity of the method and the cost of reagents are also factors in the selection of a procedure Several variables, especially the cell type to be transfected, can affect the efficiency of each of the methods discussed below and, if necessary, each technique should be optimized for a given application Each technique... manufacturer This constitutes a 2-fold excess of enzyme, for which 1 unit of activity is defined as the amount of enzyme required to digest 1 /zg of DNA at 37°C in 1 hr Simultaneous digestion with two different enzymes can be done if the digestion buffers required for each are compatible according to the manufacturer information If two incompatible enzymes are necessary, digestion with one is followed... of various techniques for analyzing Ras proteins and their functions can be found in Refs 27 and 31 However, many assays for Ras function, such as for anchorageindependent growth or migration, require a population of cells that are all expressing the protein of interest, so stable expression in selected cells is required Stable Transfection Forty-eight hours after transfection, medium is aspirated from... transfection by calcium phosphate precipitation of DNA, (2) transfection by lipid-DNA complexing, (3) infection by retrovirus, and (4) electroporation Of these, we have found the first three to be satisfactory for all our applications (Retroviral infection of mammalian cells is discussed separately below.) To maximize the desired biological readout and reduce the need for large amounts of DNA, the efficiency... comparisons, despite their ability to promote strong activity from H- and N-Ras proteins For focus-forming assays, pZIP is clearly the preferred vector, although pCGN is also effective at generating foci pDCR is intermediate in transformation assays, while pEFGP reduces the transforming ability of Ras variants, especially K-Ras, in this assay Both pBABE and pcDNA3 can generate foci with H- and N-Ras constructs,... to transfection, retroviral infection of mammalian cells offers a number of advantages First, infection is generally much more efficient in delivering DNA to cells This results in a higher percentage of cells expressing the desired protein, commonly 70-90% of a population when viral particle number is not the limiting factor in infection For transient assays, achieving a higher percentage of cells... should be confirmed by a different method Stable versus Transient Transfection Any of the described methods can be used for stable or transient transfection of cells If cells stably expressing the protein of interest are not needed, then after transfection the cells are simply subjected to whatever treatment is appropriate for the assay being performed (i.e., cells may be lysed, fixed, and stained,... even if specific antibodies for a novel protein are not available Also, the expression levels of different proteins containing the same tag can be directly compared without having to determine the relative sensitivities of two different protein-specific antibodies Antibodies to such tags can also be used to immunoprecipitate proteins and their associated complexes, or to affinity purify proteins for . link signaling from one family member to another, as well as the use of shared regulators and effectors by different family members. Concurrent with the expanded complexity of Ras family biology,. of chapters. In particular, we hope these volumes will provide valuable references and sources of information that will facilitate the efforts of newly incoming researchers to the study of. number of non-Raf effectors also mediate Ras family protein function. Third, it is increasingly clear that the cellular functions regulated by Ras go beyond regulation of cell proliferation, and