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2.3.1 Preparation of whole cell lysates

Cells were washed twice with PBS and resuspended in between 20-500|il of SDS- PAGE sample buffer (2% (w/v) SDS, 62.5mM Tris-HCl pH7.5, 10% (v/v) glycerol, ImM DTT, 0.01% (w/v) bromophenol blue). The sample was heated at 95°C for 5 min and analysed by SDS-PAGE.

2.3.2 Triton X-100 lysis of cells

Cells were washed twice with PBS and resuspended in 0.5 ml of ice-cold extraction buffer ( 50 mM Tris.HCl pH 7.4, 150mM NaCl, 50mM NaF, 5mM EDTA, 1% (w/v) Triton X-100, 500|iM sodium orthovanadate, 2mM PMSF, and 100 kallikrein inhibitor units of aprotinin). The lysate was incubated on ice for 20min, followed by centrifugation at 10,000g for 20min at 4°C to remove cell debris. The lysate was then subjected to further biochemical analyses. For rat brain and liver extracts the tissue homogenate was first centrifuged for lOmin at 10,000rpm then lysed in ice-cold extraction buffer. Following incubation on ice, the lysate was recentrifuged for 45min at 50,000 rpm at 4°C.

2.3.3 Immunoprécipitations of proteins

Cell lysates were prepared as described in section 2.3.2 and incubated with appropriate primary and secondary antibodies in tubes on a rotating wheel for 2 hours at 4°C. The immune complexes were collected by the addition of either Protein A Sepharose CL4B or Protein G Sepharose Fast flow (Pharmacia). The immunoprecipitates were collected by low speed centrifugation (2000g) and washed twice with 1ml ice-cold lysis buffer.

The samples were washed as required for further analyses or SDS-PAGE sample buffer was added to immobilised immunoprecipitates, followed by heating at 95°C for 5min and electrophoresis on an SDS-PAGE gel.

2.3.4 SDS polyacrylamide gel electrophoresis (PAGE)

SDS PAGE was performed by a procedure modified from Laemmli (1970). Gels were 0.75mm thick and comprised a 375mM Tris HCl pH8.8 resolving gel with an acrylamideibisacrylamide ratio of 30:0.5 and a 125mM Tris HCl pH 6.8 stacking gel with an acrylamideibisacrylamide ratio of 30:0.75. Electrophoresis was carried out in electrophoresis buffer (200mM glycine, 25mM Tris-HCl pH 8.3, 0.1 % (w/v) SDS), typically at 40-100V.

2.3.4.1 Coommassie Blue Staining of SDS-PAGE gels

The presence of proteins in SDS-PAGE gels was visualised by soaking the electrophoresed gel in Coomassie blue stain (45% (v/v) methanol, 10% (v/v) acetic acid, 0.25% (w/v) Coommassie blue) for 20min , followed by destaining in 20% (v/v) methanol, 14% (v/v) acetic acid. The gel was then dried under vacuum for 40min at 80°C.

2.S.4.2 Silver Staining of SDS-PAGE gels

Silver staining of SDS-PAGE gels was used to visualise amounts of protein in the nanogram range. Following electrophoresis, the proteins were fixed in the gel for Ihour using 25% (v/v) propan-2-ol, 10% (v/v) acetic acid, followed by incubation in 10% (v/v) glutaraldehyde for 30min. The gel was then repeatedly washed in water over a period of 2 hours before being sensitised in 5}ig/ml DTT. This was followed staining in 0.1%(w/v) AgNOg for 30min. The gel was then washed briefly in water then 3% sodium carbonate/0.05% (v/v) formaldehyde was added to develop the bands. Once protein bands were visible, the reaction was terminated by the addition of 25ml of 2.3M sodium citrate.

2.3.5 Estimation of protein concentration

Protein concentrations were estimated in solution using a Coommasie Blue based assay (Pierce) according to the manufacturer’s instructions. Briefly, 0.75ml of reagent was mixed with 0.75ml protein solution and the absorbance measured at 595nm. The protein concentration was then determined by comparison with a bovine semm albumin standard curve.

2.3.6 Electroblotting SDS-PAGE-resolved proteins

Following electrophoresis, separated proteins were electrophoretically transferred onto an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore Corporation) in 50mM Tris, 380mM glycine, 0.1% SDS and 20% methanol at 0.95 A for 1 hour or at 25V for 16 hours. The polyvinyldine difluoride (PVDF) membrane was pre-soaked in methanol, then washed with water and transfer buffer prior to use.

2.3.7 Western blotting

All western blot analysis was performed using enhanced chemiluminescence (ECL) reagents (Amersham Life Sciences). All incubations were performed on an orbital shaker and at room temperature unless otherwise specified. PDVF membranes were blocked for 1 hour in 5% (w/v) low fat milk powder (Marvel, Premier Brands UK Ltd.) in PBST (20mM phosphate pH7.4, 150mM NaCl, 0.05% Tween 20), then incubated with the primary antibody diluted in PBST containing 3% (w/v) low fat milk powder for 2 hours. Unbound primary antibody was removed by rinsing twice and washing (3x5min) in the same buffer, then the blots were incubated for 1 hour in the appropriate horseradish peroxidase conjugated secondary antibody (1/1000 dilution) (Pierce and Warriner (UK) Ltd) in PBST. The blots were rinsed in PBST buffer, developed with ECL reagents (Amersham Life Sciences) and autoradiographed upon x- omat XAR film, as directed in the manufacturer’s instructions.

2.3.7.1 Stripping and Reprobing PVDF membranes

Membranes were stripped of bound antibodies by immersing the membrane in stripping buffer (lOOmM 2-mercaptoethanol, 2% SDS, 62.5mM Tris-HCl pH6.7). Following incubation at 65°C for 30min with occasional agitation, the membranes were washed in large volumes of PBST to remove the mereaptoethanol and blocked Membranes were then blocked and reprobed (as described in section 2.3.7).

2.4.1 Purification of native p85a

2.4.1.1 Preparation of phosphotyrosine affinity matrix

lOmls of Actigel (Sterogene) was equilibrated in lOOmM phosphate buffer pH 7.8. O- phospho-L-tyrosine (Sigma) (50mgs) was dissolved in 50mls of the same buffer and coupled to Actigel at 4°C for 4-6hrs. The resin was washed, then unreacted sites were blocked using lOOmM Tris pH 8.0 for a further 2-4hrs. The affinity matrix was

washed several times in PBS (20mM phosphate pH 7.4, 150mM NaCl) and stored at 4°C with 0.02% sodium azide.

2.4.1.2 Phosphotyrosine Affinity Purification

The method described here utilises the inherent affinity of the two SH2 domains of p 8 5 a for phosphotyrosine (Koegl et a i, 1994), and was therefore applicable to purifying p85a or any SH2-domain containing protein, provided that the SH2 domain was not blocked due to being involved in another inter- or intra-molecular interaction.

Between 5x10^ and 2x10^ Sf9 cells were infected with a predetermined volume of baculovirus encoding p85a so as to give a multiplicity of infection (moi) of between 5 and 10. Cells were harvested 2.5 days post infection, by centrifugation at 800 g for 20min, washed in ice-cold PBS and then re-pelleted by centrifugation. The Sf9 cell pellet was resuspended in FPLC buffer A (lOmM HEPES pH7.5, 5mM NaF, 0.2mM sodium orthovanadate, 2mM EDTA, lOmM Benzam idine, 50mg/ml phenylmethylsulfonyl-fluoride [PMSF] and 5mM DTT), then the cell membranes disrupted by dounce homogenisation. After 30min incubation on ice, the lysate was cleared of particulate material by centrifugation at 35,000rpm for 45min at 4°C. All subsequent procedures were carried out at 4°C. The supernatant was loaded onto a 10ml phosphotyrosine affinity column containing 5mg phosphotyrosine per ml of matrix that had been pre-equilibrated in FPLC buffer A. The column was washed with FPLC buffer A until elution of unbound material was no longer detected by absorbance at 280nm. Bound protein was eluted at a flow rate of 2ml/min, and 2.5ml fractions were collected. Using SDS-PAGE, all fractions were assayed according to procedures described in section 2.3.4. The fractions containing p85a were pooled and buffer exchanged into 20mM Tris pH8, 5mM DTT and immediately applied on to a 4.6x100mm Poros 20 HQ column (PerSeptives Biosystems). After washing in the same buffer, bound protein was eluted with 30ml 20mM Tris pH8, 5mM DTT followed by a 30ml linear gradient from 0 to 0.5M NaCl at a flow rate of 2ml/min. Fractions (2.5ml) containing p85a were exchanged into storage buffer (50% ethylene glycol, 40mM Tris, lOmM EDTA, lOmM benzamidine and 5mM DTT) by using prepacked Sephadex 025 columns (PDIO, Phannacia) then concentrated using Centriprep microconcentrators (Amicon) with a membrane that had a molecular weight cut off of lOkDa. Purified and concentrated protein was stored at -20°C.

2.4.2 Purification of p85 mutants

Mutants of p85a with two SH2 domains were purified using the method just described (section 2.4.1.2) however, in some cases anion exchange chromatography was not used, as affinity purification gave adequate levels of purity for biochemical analyses.

2.4.5 Preparation of glutathione S-transferase-fusion proteins

Glutathione S-transferase (GST) fusion proteins were expressed using the pGEX expression vectors (Pharmacia) in E.coli. Subcloning of the desired p85a mutant in frame with the ATG of GST allowed expression of a fusion protein with a thrombin cleavage site just carboxy terminal to the GST. GST fusion proteins were purified by a modification to the procedure previously described (Smith and Johnson, 1988). Bacteria were lysed in lysis buffer (50mM Tris, 1% TritonX-100, 150mM NaCl, ImM EDTA, and ImM NaF) containing lOpg/ml leupeptin and lOpg/ml pepstatin. The GST fusion proteins were purified using glutathione-Sepharose-4B (Pharmacia), and eluted using lOmM glutathione according to the manufacturer’s instructions. Where required, proteins were cleaved with thrombin (Sigma chemicals) to remove the GST portion. Both the GST fusion proteins and the cleaved proteins were dialysed into 50% glycerol, 20mM Tris pH 8 , 150mM NaCl and ImM DTT and stored at -20°C.