Sea block buffer

Protein containing samples (equivalent of 5 × 10⁶ cells) were loaded onto a 4–15% precast Criterion polyacrylamide gel (Biorad). The separated proteins were transferred onto PVDF membranes (Millipore), and then blocked for 1 h at room temperature in a 1:1 1XPBS:SEA Block buffer (Thermo Scientific). The PVDF membranes were then incubated with primary antibodies against GRB2 (clone 23, Santa Cruz Biotechnology), LAT pY226 (clone J96-1238.58.93, BD Pharmingen), LAT pY132 (Genetex), SLP-76 pY128 (clone J141-668.36.58, BD Pharmingen), ERK1/ERK2 pY187/pT185 (Invitrogen), p38 pT180/pY182 (Cell Signaling), JNK pT183/pY195 (Cell Signaling), Akt pS473 (clone-14-6, Invitrogen), Src pY416 (Cell Signaling), pY783 PLC-γ1 (Cell signaling), PLC-γ1 (Cell Signaling), lymphocyte-specific protein tyrosine kinase (LCK) (Cell Signaling), pY (4G10, Millipore), Gsk3αβ pS21/9 (Cell Signaling), actin (clone C4, Millipore), or GAPDH (Meridian Life Sciences). Secondary antibodies conjugated to IRDye 800CW or IRDye 680 were diluted in SEA Block and incubated with the PVDF membranes for 30 min at room temperature. The membranes were then visualized using the Licor Odyssey Infrared detector.

Proteins were coated onto immulon 2 high bind 96-well microtiter EIA plates (Thermo Fisher Scientific) at 2 µg ml⁻¹ by incubating at 4 °C in 100 µl per well of coating buffer (50 mM Na₂CO₃, 50 mM NaHCO₃, pH 9.6) overnight. Empty well controls contained coating buffer only. Plates were washed in 300 µl per well of PBS containing 0.05% Tween-20 (PBST). Wells were blocked with 200 µl for 2 h at 37 °C in a humidified atmosphere. Assays using goat serum were blocked in PBST containing 5% bovine milk; assays using human serum were blocked in 50% PBST/50% SEA block buffer (Thermo Fisher Scientific). Following the blocking step, plates were incubated with 100 µl per well of serum (1:100) in PBST for 1 h at 37 °C in a humidified atmosphere. Plates were washed and incubated with 100 µl per well of a biotin-labeled, species-specific primary antibody against IgG (KPL) at 1:50,000. Plates were washed and incubated with 100 µl per well of peroxidase-labeled streptavidin (KPL) at 1:2000 for 1 h each at 37 °C in a humidified atmosphere. Detection was achieved through addition of 100 µl per well of ABTS substrate (KPL). Reactions were stopped with 100 µl per well of 1% SDS and absorbance measured at 405 nm using an ELx800 microplate reader (BioTek).

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).