Materials and methods 9

p 2 p]p |p counts obtained for each sample were normalised to the specific activity of the [y_32p]ATP used for the assay, and are expressed as fmol of PIP per min.

p70S6k assays and processing of kinase assays

After stimulations as indicated cells from every cell line used were lysed in lysis buffer 2. For 293 cells 100 pg total protein extract, and for Kit225 cells cell extracts corresponding to 3 x 10® cell equivalents (non-transfected cells) or 1.5 x 10® live cell equivalents (transfected cells) were used per point.

The immunoprecipitates were washed three times in lysis buffer 2, and once in

p70S6k assay buffer (50 mM Mops pH7.2, 5 mM MgCl2, 0.1% Triton X-100) and

assayed as described (Ming et al., 1994). Briefly, the reaction was initiated by the addition of 10 pi p70S6k reaction bu/'/er containing 3 pCi [y-®2p]ATP, 100 pM ATP, 50 mM Mops pH7.2, 5 mM MgCl2, 10 mM pNPP, 1 mM dithiothreitol (DTT), 500 nM Protein

Kinase Inhibitor (PKI) (Sigma), 0.05% Triton X-100 and 20 pg 408 ribosomes as a source for 86 substrate (a gift from George Thomas, FMI, Basel). After 30 min at 37 °C the reaction was terminated by adding reducing 8D8-PAGE sample buffer and boiling. Proteins were resolved by 8D8-PAGE. The lower part of the gel was stained with Coomassie Blue, destained in 30%(v/v) methanol/10%(v/v) acetic acid, dried and ®2p- labelled 86 proteins were detected by autoradiography. The levels of p7086k protein in each immunoprecipitate were assessed by transferring the proteins in the upper part of the gel onto PVDF membranes and Western blot analysis with 12CA5 mAbs or M l Abs using the ECL detection system (Amersham). If the p7086k protein levels in the immunoprecipitate were not equal, activities were normalised for p7086k expression levels by quantitation of Western blots probed with M l Abs followed by ^^®l- conjugated protein A (Amersham). Quantitation of incorporated ®2p into 86 or of bound ■'25|-conjugated protein A was performed using a Phosphorlmager.

C h a p te r 2 Materials and methods 9 1

PKB assays

After stimulations as indicated cells from every cell line used were lysed in lysis

buffer 3 (120 mM NaCI, 50 mM Hepes pH7.4, 10 mM NaF, 1 mM EDTA, 40 mM 13-

glycerophosphate pH7.5, 1% NP40, 0.1 mM PMSF, 0.1 mM Na3V0 4). For Kit225 cells

cell extracts corresponding to 3x10® cell equivalents (non-transfected cells) or 1.5 x 10® live cell equivalents (transfected cells) were used per point. For Jurkat cells cell extracts corresponding to 1 x 10® live cell equivalents were used per point.

The immunoprecipitates were washed twice in lysis buffer 3, twice in high salt

wash buffer {500 mM LiCI, 100 mM Tris pH7.5, 1 mM EDTA pH7.5) and once in PKB

assay buffer {50 mM Tris pH7.5, 10 mM MgCl2, 1 mM DTT). The reaction was initiated

by the addition of 15 pi PKB reaction bu/fier containing 3 pCi [y-®^P]ATP, 50 pM ATP, 7.3 mM MgCl2, 730 pM DTT, 500 nM PKI, 40 mM Tris pH 7.5, and 2.5 pg histone 28 (H2B)

(Boehringer Mannheim). After 30 min at 25 °C the reaction was terminated by adding reducing SDS-PAGE sample buffer and boiling. Proteins were resolved by SDS-PAGE and the gel was treated as for p70S6k assays. To detect PKB proteins Western blot analysis was performed with Rac-PK-CT Abs.

Erk assays

Cells and cell extracts were processed as for p70S6k assays except that for 293 cells 250 pg total protein extract was used.

Precipitated immune complexes were washed three times with lysis buffer 2 and once with Erk wash buffer (30 mM Tris pHS.O, 20 mM MgCl2, 2 mM MnCl2). The

reaction was initiated by the addition of 10 pi Erk reaction buffer containing 4 pCi [y- 32p]ATP, 20 pM ATP, 20 mM MgCl2, 2 mM MnCl2, 5 mM pNPP, 500 nM PKI, 30 mM Tris

pH8.0, and 15 pg Myelin Basic Protein (MBP) (Sigma). After 30 min at 37 °C the reaction was terminated by adding reducing SDS-PAGE sample buffer and boiling. Proteins were resolved by SDS-PAGE and the gel was treated as for p70S6k assays. To detect Erk proteins Western blot analysis was performed with 12CA5 mAbs as

C hapter 2 Materials and methods 9 2

primary Ab, rabbit anti-mouse IgG as secondary Ab (DAKO) and ‘'25|-conjugated protein A.

Jnk assays

Cells and cell extracts were processed as for p70S6k assays. For Jurkat cells cell extracts corresponding to 1 x 10® live cell equivalents were used per point.

Precipitated immune complexes were washed three times with lysis buffer 2 and once with Jnk assay buffer (25 mM Hepes pH7.4, 20 mM (3-glycerophosphate pH7.0, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM Na3V0 4 ). The reaction was initiated by the

addition of 15 p,l Jnk assay buffer containing 4 (xCi [y-®^P]ATP, 20 pM ATP, 333 nM PKI and 2.5 pg GST-Jun (aa 1-135) (Kyriakis et al., 1994). After 20 min at 30 °C the reaction was terminated by adding reducing SDS-PAGE sample buffer and boiling. Proteins were resolved by SDS-PAGE and the gel was treated as for p70S6k assays. To detect Jnk proteins Western blot analysis was performed with JNK2 (FL) Abs.

Protein expression analysis

To test for effector protein expression in transfected cells, postnuclear cell extracts corresponding to 2 to 4 x 10® cell equivalents were analysed. Postnuclear cell lysates were acetone-precipitated for Western blot analysis as follows: 700 pi pre­ cooled (-20 °C) acetone was added to 500 pi lysate and samples were incubated at -20 °C for 2 h. Precipitated proteins were pelleted in an Eppendorf microcentrifuge at 15,000 X g for 30 min at 4 °C. The supernatant was discarded and the protein pellets were washed once with 400 pi pre-cooled (-20 °C) ethanol. The proteins were dried and rehydrated by adding 20 pi to 40 pi distilled water and incubating for 15 min at 37 °C. An equal volume of two times reducing sample buffer was added and samples were boiled for 8 min prior to SDS-PAGE as detailed in a following section.

To test for effector protein expression in transfected 293 cells, 50 pg postnuclear cell extract were mixed with reducing sample buffer and analysed.

Chapter 2 Materials and methods 9 3

To test for the presence of proteins when proteins were affinity purified, the protein precipitates were washed four times in lysis buffer. With the final wash the immunoprecipitates were transferred into a separate tube and the rest of the solution was removed using a Hamilton syringe. Samples were boiled for 8 min in 60 pi to 90 pi reducing sample buffer prior to SDS-PAGE as described below.

SDS-polyacrylamide gel electrophoresis (PAGE)

For a comprehensive description of SDS-PAGE see 'Gel electrophoresis of Proteins' (Names et al., 1981). The principal of discontinuous SDS-PAGE used here was first described by Laemmli for acrylamide capillary tube gels (Laemmli, 1970).

The reducing sample buf/ier contained: 3%(w/v) SDS, 10%(v/v) glycerol, 62.5

mM Tris-HCI pH6.8, 5%(v/v) p-mercaptoethanol, 0.005%(w/v) bromophenol blue. For normal gels a readily made stock solution of 30%(w/v) acrylamide/0.8%(w/v) N,N'- methylene-bis-acrylamide (National Diagnostics) was used for 7-17% gradient, 7%, 8%, 10% or 12.5% SDS-polyacrylamide gels (percentage acrylamide for running gel (pH8.8)). For shift gels a Cold Spring Harbour gel mix containing a lower concentration of N,N'-methylene-bis-acrylamide was used (10% acrylamide/0.13% N,N'-methylene- bis-acrylamide or 15% acrylamide/0.075% N,N'-methylene-bis-acrylamide). The stacking gel generally had a concentration of 5% acrylamide (pH6.8). The running buffer

used contained: 25 mM Tris, pH8.3, 190 mM glycine, 3.5 mM SDS. Prestained protein molecular weight standards had a molecular weight range of 14,300 to 200,000 and were purchased from GI BOO BRL

Western blot analysis

After SDS-PAGE, samples for Western blot analysis, either immunoprecipitates or acetone-precipitated cell lysates, were transferred by wet electroblotting onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore) (Kamps and Sefton, 1989). The transfer buffer used was 10 mM CAPS buffer (Sigma), p H I I , and

Chapter 2 Materials and methods 9 4

blotting was carried out for 8 h to 14 h at 0.3 A. The filters were blocked using 5% dried skimmed milk (DSM) in PBSA. The membranes were then incubated overnight at 4 °C with the first layer antibody in a sealed plastic bag on a rocker. The antibodies were applied typically in PBSA containing 0.5% DSM, 0.05% Tween-20, 0.1% NaNa in the following concentration or dilution:

1.7 pg/ml 1 pg/ml 0.5 pg/ml 0.5 pg/ml 1 pg/ml 0.25 pg/ml 1 pg/ml 2.5 pg/ml 1/100 1/1,000 1/500 1/500 1/5,000 0.5 pg/ml 0.1 pg/ml ant ant ant ant ant ant ant ant ant ant ant ant ant ant ant

-p85a , U5 (mouse); 5% DSM, 0.5% Tween-20, 0.1% NaNg -rCD2, 0x34 (mouse)

-Myc epitope, 9E10 (mouse) -HA epitope, 12CA5 (mouse) -phosphotyrosine, 4G10 (mouse) -Grb2, Grb2 (mouse)

-Ras, pan-ras (OP41) (mouse) -m S osI, Sosi (rabbit)

-m S osI, Sos3 (rabbit) -Ick, RNGS (rabbit)

-fyn, anti-yes, CST-1 (rabbit) -pi 10, p i 10 (PW38) (rabbit) -p70S6k, M5 (rabbit)

-PKB, Rac-PK-CT (rabbit) -Jnk2, JNK2 FL (rabbit)

For far-Western blotting, 1 pg/ml Myc-tagged fusion proteins were incubated overnight in PBSA containing 1% DSM, 0.1% Tween-20, 0.1% NaNs. Membranes were washed three times for 15 min at room temperature in PBSA containing 1% DSM, 0.1% Tween-20 and incubated with 9E10 mAbs for 2 h at room temperature in this buffer.

The membranes were washed three times in PBSA containing 0.05% Tween- 20 and proteins were visualised using a chemiluminescence detection system (ECL, Amersham) with a sheep anti-mouse Ig or donkey anti-rabbit, horseradish peroxidase as secondary antibody (Amersham). Secondary antibodies were applied in a 1/5,000 to 1/10,000 dilution in PBSA containing 0.05% Tween-20.

Chapter 2 Materials and methods 9 5

Identification of unknown proteins by mass spectrometry

The Identification of unknown proteins by mass spectrometric analysis is based on a combination of protease digestion, matrix-assisted laser-desorption ionisation mass spectrometry (MALDIMS) and screening of peptide-mass databases, e.g. the molecular weight search (MOWSE) peptide-mass database at the SERC Daresbury Laboratory, UK (Pappin et al., 1993). This method generates molecular weight fingerprint maps and allows an identification of proteins from as few as three or four experimentally determined peptide masses when these are screened against a fragment database that is derived from over 160,000 proteins (Pappin et al., 1993; Pappin et al., 1996). The analysis requires very low amounts of protein and the sensitivity extends into the low fentomole range. Additional information about the identity of a protein can be gained by specific chemical modification (e.g. estérification of acidic residues) of the sample followed by a comparative search with these parameters in the MOWSE database that includes search options for these criteria.

Proteins isolated from T cell lysates were separated by SDS-PAGE and transferred onto PVDF membrane. The membrane was washed thoroughly in deionised water to remove buffer salts and dried in vacuo ior 20 min. Proteins were visualised by staining with sulforhodamine B (Kodak Ltd.) as described (Coull and Pappin, 1990). Digestion of proteins: The apparent protein bands were excised from the membrane, dried, placed in 0.5 ml eppendorf tubes and wet with 2-4 pi of 50 mM ammonium bicarbonate solution containing 1%(w/v) octyl glucoside and 40 ng/ml trypsin protease (modified grade, Promega). Digestion and processing of samples were performed as described (Sutton et al., 1995). Aliquots of the sample (0.3-0.5 pi) were applied to sample slides, and dried under high vacuum for 30 min for mass spectrometry analysis. Estérification of peptides: Aliquots of the sample (0.5-2 pi) from the digested protein were dried in vacuo and treated with 10 pi of 1%(w/v) thionyl chloride in dry methanol for 30 min at 50 °C to effect estérification (methyl esters) of peptide acidic residues (E=Glu,

Chapter 2 Materials and methods 9 6

D=Asp). Samples were processed as described (Pappin et al., 1996) and aliquots were dried onto target slides for mass spectrometry analysis.

Mass spectrometry analysis of samples: Dry samples on target slides were re-wet with 0.5 \l\ matrix solution (1%(w/v) alpha cyano-4-hydroxycinnamic acid in 50% aq. acetonitrile containing 0.1% TFA and 200 fmol/jiil insulin B chain as internal standard), allowed to air dry and analysed by MALDIMS using a Finnigan MAT LaserMat 2000 mass spectrometer (Mock et al., 1992). Spectra were calibrated using the insulin B chain as an internal standard. Observed proteolytic fragment masses were screened against the MOWSE peptide-mass database (established by Darryl Pappin in collaboration with the SERC Daresbury Laboratory, Warrington, UK) as described (Pappin et al., 1993; Pappin et al., 1996).

Gene expression analysis

Determination of chloramphenicol acetyltransferase (CAT) activity was determined according to the method of Sleigh (Sleigh, 1986).

NIH 3T3 cells: Eight to 10 hours after inductions as indicated transfected serum- starved NIH 3T3 cells were washed with PBSA and cells were lysed in 150 pi C A T

lysis buffer 1 (0.65% Triton-XlOO, 10 mM Tris pHB, 1 mM EDTA, 150 mM NaCI). The

relative protein concentration of each postnuclear lysate was measured. An equivalent amount of protein was used per CAT assay, sample volumes were kept constant (typically 75 pi). Samples were heat-inactivated for 10 min at 68 °C and cooled on ice. The CAT reaction was initiated by adding 40 pi of CAT assay reaction buffer (21 mM [^^Cjacetyl coenzyme A (0.05 pCi), 0.5 mM acetyl coenzyme A, 5 mM chloramphenicol, 500 mM Tris pH8.0). After 2 h to 6 h at 37 °C reactions were terminated by freezing. Acetylated chloramphenicol was extracted with 200 pi ethylacetate and vortexing vigorously. The phases were separated by centrifugation in an Eppendorf microcentrifuge at 15,000 x g for 5 min and 100 pi of the upper, organic phase was removed to a scintillation vial containing 3 ml EcoLume™ scintillant (ICN) and counted

Chapter 2 Materials and methods 9 7

using a calibrated ^^C-label program on a Beckman LS6000 series scintillation counter. Transfection efficiency was monitored with appropriate p-galactosidase (lacZ) expression vectors as described (Price et al., 1995). The data are presented as the ratio of CAT activity (percentage conversion) to p-galactosidase optical density (OD) units.

T cells: Fourteen to 16 hours after inductions as indicated Kit225 or Jurkat T cells were harvested and cells were lysed in 200 pi CAT lysis buffer 2 {0.65% NP40, 10 mM Tris pH8 , 1 mM EDTA, 150 mM NaCI). Similar cell equivalents were tested for enzymatic

activity able to transfer ^^C-labelled acetyl groups onto chloramphenicol as described above. The data are presented as percentage conversion.

Minipreparation of piasmid DNA

Single colonies were used to inoculate 5 ml of Luria broth (L-broth) (10 g bacto tryptone, 5 g yeast extract, 10 g NaCI, ad 1 I) containing 50 pg/ml ampicillin (or other appropriate antibiotics). Minipreps were carried out using 3 ml of an E. coli culture grown for 8 h to 15 h. Cells were pelleted by centrifugation and the pellet was resuspended in

200 pi solution I (25 mM Tris pH8, 50 mM Glucose, 10 mM EDTA) on ice. 400 pi of

solution II (0.2 M NaOH, 1% SDS) were added and the tubes were inverted 3 to 4

times. After addition of 300 pi of solution III (60%(v/v) 5 M potassium acetate, 11.5%(v/v) glacial acetic acid, in water), the suspension was mixed and left on ice for 15 min. The sample was spun in a microcentrifuge for 5 min and the supernatant was transferred to a new tube. The plasmid DNA was precipitated with 600 pi isopropanol (2-propanol) at 4 °C. After 10 min centrifugation, the dried pellet was resuspended in 40 pi H2O and 5 pi to 10 pi were used in restriction digest analysis. In some experiments,

the DNA solution was further purified with an equal volume (typically 100 pi) of 5 M LiCI to precipitate high molecular weight RNA. After incubation on ice for 5 min, RNA was pelleted and the plasmid DNA in the supernatant was precipitated with 2 to 3 volumes ethanol at -20 °C.

Chapter 2 Materials and methods 9 8

Maxipreparation of piasmid DNA

Milligram quantities of very pure plasmid DNA for cell transfections and microinjections were obtained by a alkaline/SDS lysis method (Birnboim and Doly, 1979) followed by isopycnic centrifugation in a caesium chloride solution (Radloff et al., 1967). Briefly, 400 ml culture of E. co//carrying the desired plasmid, grown overnight in

brain heart infusion (BHI) broth (37 g BHI powder in 11), were pelleted at 4,000 rpm, and

resuspended in 20 ml solution I. Bacterial cells were lysed by adding 40 ml of solution II. Denatured proteins, chromosomal DNA and cellular debris were precipitated by adding 30 ml of solution III. After 15 min on ice, precipitates were removed by centrifugation, the DNA in the supernatant was recovered with 70 ml isopropanol at 4 °C and the pellet was washed with 10 ml of cold 75% ethanol. The DNA pellet was resuspended in 4.5 ml of TE buffer {^0 mM Tris pH8, 1 mM EDTA) and 4.9 g CsCI was added. After the

CsCI was dissolved 0.4 ml of 1 0%(w/v) ethidium bromide solution was added, the

tubes were covered with aluminium foil and the solution was incubated for 30 min at room temperature to precipitate proteins. The proteins were pelleted by centrifugation at 10,000 rpm (Beckman JA-20 rotor) for 20 min at 20 °C. The supernatant was transferred to a heat-sealable ultracentrifugation tube (Beckman) and centrifuged in a vertical rotor (VTi 65.2, Beckman) at 65,000 rpm for 4 h at 20 °C to allow density gradient formation. The plasmid band was harvested with a syringe, transferred to a new heat-sealable ultracentrifugation tube and the plasmid DNA was further purified on a second CsCI gradient. The plasmid DNA was removed and the ethidium bromide extracted with water saturated n-butanol. The DNA was precipitated with 2 to 3 volumes of ethanol and resuspended in TE buffer.

Restriction enzyme anaiysis of DNA

Plasmid DNA (1 pg) was digested in a volume of 10 pi in the buffer supplied by the manufacturer at 37 °C for 30 min to 60 min using -1 0 units (1 pi) of restriction enzyme. Fragments were separated on an agarose gel.

Chapter 2 Materials and methods 9 9

Partial digest of DNA

For cloning purposes restriction sites had to be used that occurred more than once in the cDNA. Hence digest were carried out under limiting conditions in the presence of 0.05 |ig/)il ethidium bromide, less than 1 unit of restriction enzyme (< 0.1 pi enzyme) per 1 pg DNA (10 pi volume), short incubation times or a combination of these conditions.

Phosphatase treatment of DNA

To avoid religation of linerised vector, 5'-terminal monophosphate groups of DNA were hydrolysed with 1 unit of alkaline phosphatase (New England Biolabs) per pmol DNA ends in phosphatase buffer (50 mM NaCI, 10 mM Tris pH8, 10 mM MgCl2, 1

mM DTT) for 30 min at 37 °C (typically 0.5 pg DNA/10 pi). EDTA (pH8) was added to 5

mM and the enzyme was heat-inactivated for 10 min at 75 °C. The solution was extracted with an equal volume of phenol/chloroform and the DNA precipitated with 0.1 volume 3 M sodium acetate pH5.5 and 2 volumes ethanol and resuspended in TE buffer.

Agarose gei electrophoresis

For fragments between 500 and 1,500 basepairs, an agarose gel of 1% was used. Fragments larger than 1,500 basepairs were separated on a 0.7% gel. Ultra pure agarose (GIBCO) was boiled in 1 x TAE buffer {0.04 M Tris-acetate, 2 mM EDTA pH8;

50 X TAE buffer: 242 g Tris, 57.1 ml glacial acetic acid, 100 ml 0.5 mM EDTA pH8, ad 1 I),

ethidium bromide (0.5 pg/ml) was added and the solution was left to solidify in a gel plate. The DNA was loaded with 0.1 volume of 10 x DNA sample buffer (40% glycerol, 0.4% bromophenol blue) and electrophoresed at 120 to 150 mAmp.