Anti perk e 4

Cell lysates were prepared with the ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Burlington, Canada). Twenty µg of the cell lysate isolated from Rin-5F or islet cells or 20 µg proteins precipitated from the cell culture medium after cell treatment were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblot analysis was performed using anti-PARP-1 (H-250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PAR (H10; Alexis Biochemicals, San Diego, CA, USA), anti-Akt 1/2/3 (H-136; Santa Cruz Biotechnology), anti-pAkt 1/2/3 (Ser-473-R; Santa Cruz Biotechnology), anti-ERK 1/2 (K-23; Santa Cruz Biotechnology), anti-pERK (E-4; Santa Cruz Biotechnology) antibodies. Blots were probed with horseradish peroxidase-conjugated secondary antibodies. Staining was performed by the chemiluminescent technique according to the manufacturer's instructions (Amersham Pharmacia Biotech, Amersham, UK). If different set of samples were run on different gels, immunoblot analysis was performed in the same time, under the same conditions. All membranes were exposed under the same film and thus exposure and development did not influence the obtained results.

Immunoprecipitation was carried out with cell lysates (200 µg) obtained after lysis in 50 mM Tris 7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 1 mM PMSF, 1 µg/mL aprotinin, and 1 µg/mL leupeptin (lysis buffer). Cell lysates were incubated for 2 h on ice with 0.5 µg of anti-pAkt 1/2/3 (Ser-473-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pERK (E-4; Santa Cruz Biotechnology) antibodies or equal amount of control IgG (sc-2027; Santa Cruz Biotechnology). Protein A/G-coupled agarose beads (SC-2003; Santa Cruz Biotechnology) were added for 2 h at 4°C under constant agitation. The beads were pelleted and washed five times with lysis buffer. The immunoprecipitated proteins were resuspended in the sample buffer, separated by SDS-PAGE and analyzed by immunoblot analysis.

To confirm the stimulation via phosphotyrosine and phospho-ERK1/2, and to confirm expression/purification of constructs expressed in insect cells, lysates were run on SDS-PAGE gels and transferred to nitrocellulose membranes using standard methods. Antibodies used for detection: 4G10 Platinum anti-phosphotyrosine (Millipore), anti-pERK (E-4) (Santa Cruz), anti-β-actin (AC-15) (Sigma–Aldrich), anti-LAT (Upstate (Millipore)), and anti-FLAG-HRP (Sigma–Aldrich). The self-produced VAMP7 antibody was a kind gift of Dr. Andrew Peden (The University of Sheffield, UK).