Rabbit anti src pan

Western blotting was performed to analyze individual samples (biological replicates) for validation purposes. Platelets were lysed in 2% SDS (final concentration) and following protein quantitation by Pierce 660 nm Protein Assay (Thermo Scientific, USA), proteins were separated by 11% SDS-PAGE. Five micrograms of protein were loaded per lane. The primary antibodies used for immunoblotting were rabbit anti-phospho-SRC (Tyr419) (Invitrogen), dilution 1:1,000 and rabbit anti-SRC pan (Invitrogen). Following washes in TBS-T, membranes were exposed to horseradish peroxidase–labeled goat anti-rabbit (dilution 1:5,000) (Pierce, Rockford, IL), and processed using an enhanced-chemiluminiscence system (ECL, Pierce, Rockford, USA).

Western blotting was performed to analyze individual samples (biological replicates) for validation purposes. 11% SDS-PAGE gels were used, loading 10 μg of protein per lane. In some cases, 2D-western blotting validations were carried out, using IPG strips pI 4–7, 7 cm (GE Healthcare). In that case, 30 μg of protein were loaded per strip; obese and lean samples were analyzed in parallel. IEF (first dimension) was for 11Kvh; second dimension was on 11% SDS-PAGE gels.
Following electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5% BSA in TBS-T (20 mM Tris-HCl, (pH 7.6), 150 mM NaCl and 0.1% Tween 20) overnight at 4 °C and incubated for 90 min at room temperature with the following primary antibodies: rabbit anti-phospho-SRC (Tyr418) (Invitrogen), dilution 1:1000; rabbit anti-SRC pan (Invitrogen), dilution 1:1000; mouse anti-FIB (sc-69775, Santa Cruz), dilution 1:1000; mouse anti-PLCγ2 (sc-5283, Santa Cruz), dilution 1:1000; rabbit anti-GAPDH (SIGMA), dilution 1:5000; and anti-G6f (1:1000), produced by CovalAB UK (Cambridge, UK). Following washes in TBS-T, membranes were exposed to horseradish peroxidase–labeled goat anti-rabbit, or goat anti-mouse antibodies (dilution 1:5000) (Pierce, Rockford, IL), and processed using an enhanced-chemiluminiscence system (ECL, Pierce, Rockford, USA).