Western Blot Analysis of Signaling Proteins

Following SDS-PAGE, resolved proteins were transferred onto a PVDF membrane (Millipore) and then blocked for 1 h at room temperature in a 1:1 solution of SEA Block buffer (Thermo Scientific) in 1X PBS. Membranes were incubated for 1 h at room temperature or overnight at 4 °C with primary antibodies followed by two washes in 0.05% Tween-20 in 1X PBS. Secondary anti-mouse or anti-rabbit DyLight 680- or 800-conjugated antibodies were applied for 1 h at room temperature followed by 2 washes. Blots were then visualized using the Licor Odyssey Infrared detector. Densitometric analysis of protein bands was performed using Odyssey’s v3.0 software and normalized to GAPDH. The followed primary antibodies were used: GRB2 (BD Pharmingen or Cell Signaling Technology); LAT pY226 (clone J96-1238.58.93, BD Pharmingen); and GAPDH (Meridian Bioscience).