Anti mouse hrp conjugated secondary antibody sc 2005

Proteins were extracted from T cells using lysis buffer (Pepstatin, PMSF 100 mM, Sodium fluoride 1M, Sodium orthovanadate, Leup, 1% Brij 96 V solution) (Sigma-Aldrich). Protein concentration was determined by a DC Protein Assay (BioRad) according to the manufacturer’s recommendations. Western blotting was performed according to Bourdon’s (Dundee, UK) guidelines using reagents and devices from ThermoFisher Scientific. In brief, samples were loaded on a precast polyacrylamide gel (Bolt 10% Bis Tris Plus) and separated by electrophoresis. Samples were transferred to a nitrocellulose membrane (iBlot 2 Transfer Stacks) using the iBlot 2 Dry Blotting System. Membranes were blotted with the primary antibodies (listed above) overnight at 4°C and detected with anti-rabbit HRP-conjugated secondary antibody 7074S (Cell Signaling Technology, Cambridge, UK) (dilution 1:5000), anti-mouse HRP-conjugated secondary antibody sc-2005 (Santa Cruz Biotechnology, Heidelberg, Germany) (dilution 1:2000) anti-sheep HRP-conjugated secondary antibody ab7111 (Abcam, Cambridge, UK) (dilution 1:5000) for 1 hour at RT. Finally, chemiluminescence was detected with iBright Western Blot Imaging System after 5 min incubation with SuperSignal West Femto substrate (ThermoFisher Scientific) and 1 min exposure time.