ELECCIONES Y COMPORTAMIENTO ELECTORAL

2.7 . 1 Preparation of proteolytic d i g e s t s for Edman micro­ s e q u e n c i n g MALDI-TOF m a s s spectrometry

Phosphoproteins labelled with ^^P were TCA precipitated and separated using SDS- PAGE. Gels were stained with Coomassie Brilliant Blue and protein bands carefully excised with a clean scalpel, washed once in HPLC grade water and then macerated and destained in 50% acetonitrile. After vacuum drying the gel pieces were rehydrated in 10 mM Tris.HCl, pH 7.5-8.5, containing the appropriate protease at an enzyme to substrate ratio of 1:50 and digested overnight at 37®C. Peptides were extracted three times in 50% acetonitrile containing 5% trifluoroacetic acid (TFA), concentrated in a Speedvac and applied to a 1 x 10 mm C8 reverse phase HPLC column. Peptides were collected over a

linear gradient of 0-60% acetonitrile, 0.08% TFA and their radioactivity determined by Cerenkov counting. The composition of fractions corresponding to peaks of activity was determined by mass analysis using a Finnigan MALDI-TOF Lasermat instrument. Fractions containing mixtures of peptides were further separated on a C18 HPLC column and re-analysed by MALDI-TOF mass spectrometry.

2 . 7 . 2 P h o s p h o a m i n o acid a n a l y s is

HPLC fractions containing ^^P-labelled phosphopeptides were dried and subjected to acid hydrolysis in 6M HCl at 110°C for 75 min. Hydrolysates were dried overnight in a

Speedvac and phosphoamino acids were analysed by one dimensional thin layer electrophoresis as follows: samples were resuspended in 1 0 |il of electrophoresis buffer

(pyridine:acetic acid:H2 0, 10:100:1890) containing 10 mM (9-phosphoserine, O-

phosphothreonine, and 0-phosphotyrosine standards and spotted onto a Kodak cellulose chromatogram sheet which was then sprayed evenly with electrophoresis buffer. Electrophoresis was performed at 1000 V, 50 mA for 45 min with cooling, after which the plate was dried, stained with ninhydrin spray and developed for 1 minute at 110°C. The chromatogram was exposed to X-ray film and the positions of ^^P labelled phosphoamino acids correlated with the ninhydrin stained standards.

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2.8

Immunological techniques

Immunological methods not detailed here were performed according to Harlow and Lane (1988). Unless otherwise indicated in figure legends, data obtained using immunological techniques is representative of at least two such experiments.

2 .8 .1 Affinity purification of polyclonal antisera

Polyclonal antiserum was batch-purified on the L4 PtdlnsPK I la peptide immunogen immobilised on Affigel (see Section 2.5.2.d). After washing the affinity matrix in PBS,

1.5 ml antiserum was applied to a 150 pi bed volume of affinity matrix and incubated for 30 min at 4°C. The solid phase was pelleted by centrifugation and then washed extensively in PBS. Bound antibodies were eluted in 200 pi glycine.HCl, pH 2.5 which was rapidly neutralised with NaOH.

2 . 8 . 2 Western blotting

Proteins separated on SDS-PAGE gels were transferred to pre-wetted PVDF membranes (Immobilon-P, Millipore) by electrophoresis in transfer buffer (25 mM Tris.HCl, 192 mM glycine, and 20% (v/v) methanol) using an LKB Multiphor II electrophoresis apparatus. Typically, proteins were transferred for 1 h at a charge density of 0.8 mA/cm^.

After transfer the membrane was blocked for 1 h in PB ST (Ix phosphate buffered saline containing 0.1% Tween 20) containing 5% (w/v) skimmed milk powder. After washing three times for 10 min in PB ST, the membrane was incubated for 1 h in PB ST containing 0.5% skimmed milk powder and the primary antibody at the appropriate dilution. Following this step the membrane was washed again and incubated for 1 h with the secondary horse radish peroxidase (HRP)-conjugated antibody as above. The unbound secondary antibody was washed away in PB ST and specifically bound antibody detected using the Enhanced Chemiluminescence (ECL) detection reagent (Amersham).

2 . 8 . 2 Immunoprécipitation

Cells (1-10 X 10^) in suspension cultures or monolayers were washed free of serum with cold PBS before lysis on ice in 1 ml of 100 mM Tris.HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM benzamidine, 10 pg/ml aprotinin, and 10 pg/ml leupeptin. Detergent insoluble material was sedimented by centrifugation at 18,000 x g for 10 min and the supernatant incubated with protein A Sepharose or protein G-Sepharose (Pharmacia) for 30 min at 4®C. After this pre-clearing step, the supernatant was taken to fresh tubes containing 1 - 2 pg of the relevant antibody

immobilised on protein A or -G Sepharose and incubated for 1 h at 4°C. The Sepharose beads were washed at least 4 times in lysis buffer before analysis by SDS-PAGE or assayed for lipid kinase activities directly.

2 . 8 . 3 Indirect Im mu nof luo res cen ce

Cells grown on coverslips were fixed in PBS (without Ca^+ or Mg^+) containing 3.7% formaldehyde for 10 min, permeabilised with PBS (with Ca^+ or Mg2+) containing 0.2% Triton X-100 for 5 min and then washed twice with PBS. Coverslips were incubated for 45 min inverted on parafilm with 15 p,l primary antibody at 1:500 dilution (or otherwise diluted approriately) in PBS containing 0.1% (w/v) BSA. After washing 6 times in PBS,

the coverslips were incubated in a similar way with FITC-labelled secondary antibody at a 1:400 dilution, then washed six times and mounted on slides for viewing. For co-staining of the actin cytoskeleton, 0.1 pg/ml rhodamine-labelled phalloidin (Molecular Probes) was included with the secondary antibody.

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