Anti lat upstate

Western blot samples were eluted and separated by SDS-PAGE as described above, then blotted to nitrocellulose membranes using standard procedures. After blocking the membrane in 5% (w/v) fat-free milk powder in TBS-T (TBS with 0.5% Tween-20), membranes were incubated with primary antibodies overnight at 4 °C. Membranes were then incubated with secondary HRP-coupled antibodies for 1 hr at room temperature, and visualized using an Intas Advanced Imager. Primary antibodies used were rabbit polyclonal anti-LAT (Upstate (Millipore)), mouse monoclonal anti-CD229/SLAMF3 (R&D Systems), rabbit polyclonal anti-PSMF1 (gift from Prof. Kreuger), and rabbit monoclonal anti-ZDHHC18 (Abcam).

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).