Plck y394

Cellular lysates were mixed with Laemmli sample buffer, heated at 95 °C for 10 minutes and separated on NuPAGE Bis-Tris gels by electrophoresis and transferred to nitrocellulose membranes using the iBlot transfer system (Thermo Scientific). Membranes were incubated in 3% fat-free dry milk for 1 hour at room temperature followed by overnight incubation with primary antibodies. Proteins were detected with Super Signal West Dura (Thermo Scientific #34075) using a Bio-Rad ChemiDoc MP imaging system. Immunoblots were quantified using ImageJ (NIH). Primary antibodies used were: pLck (Y394, R&D Systems), pZAP-70 (Y319), AP1, NFĸB and Lck from Cell Signaling, pLAT(Y226), total ZAP-70, and NFAT-1 from BD Biosciences, total LAT from Biolegend, beta-Actin from Sigma, CD3 (OKT3), and GAPDH from Thermo Fisher, CD28 from BD Biosciences and acetyl histone H3K9 and trimethyl histone H3K27 from Millipore.

Cellular lysates were mixed with Laemmli sample buffer, heated at 95°C for 5 minutes and separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes using iBlot transfer system. Membranes were incubated in 10% fat-free dry milk for 1 hour at room temperature followed by incubation with primary antibodies. Proteins were detected with Super Signal West Dura (Thermo Scientific 34075) using a Bio-Rad ChemiDoc MP imaging system. Immunoblots were quantified using ImageJ (NIH). Primary antibodies used were: pLck (Y394, R&D Systems), pZAP-70 (Y319), total Lck and lamin A/C from Cell Signaling, pLAT(Y226), total ZAP-70, and NFAT-1 from BD Biosciences, total LAT from Biolegend, GAPDH from Invitrogen, beta-actin from Sigma and CD3 (OKT3) from Thermo Fisher.

Cellular lysates were mixed with Laemmli sample buffer, heated at 95 °C for 5 min and separated on NuPAGE Bis-Tris gels by electrophoresis and transferred to nitrocellulose membranes using the iBlot transfer system (Thermo Scientific). Membranes were incubated in previously heated 3% fat-free dry milk for 1 h at room temperature, followed by overnight incubation with primary antibodies. Proteins were detected with Super Signal West Dura (Thermo Scientific, #34075) using a Bio-Rad ChemiDoc MP imaging system. Immunoblots were quantified using ImageJ (NIH) by stripping and reprobing the same membrane for the indicated loading controls for normalization. Integrated Density (IntDen, the product of area and mean gray value) was calculated for each condition. In each experiment, the IntDen value was obtained for each condition and was normalized to the value obtained in the control stimulated LV condition using the following formula: (IntDen value/IntDen value at stimulated LV). Primary antibodies used were: pLck (Y394, R&D Systems), pZAP-70 (Y319), total Lck, total ZAP-70, total LAT and anti-HA from Cell Signaling, pLAT(Y226) from BioLegend, and β-actin and GAPDH from Sigma.