Anti α tubulin primary antibody

Cells were lysed with 200 μl of Lysis buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF). 18 μl samples were then loaded onto gels alongside 8 μl of protein ladder. The proteins were then transferred to nitrocellulose membranes before incubation in blocking buffer (5% (w/v) skimmed milk powder in TBS-T) for 1 hour at room temperature. The blots were then washed in TBS-T and incubated overnight with anti-NFATc2 (NFAT1) (BD Transduction Laboratories) or anti-phospho-p65 (S536) (Cell Signaling Technologies) primary antibodies (BD Transduction Laboratories) at 1:5000 and 1:1000 dilution respectively, in 5% (w/v) BSA in TBS-T. Blots were washed in TBS-T and then incubated for a further hour with a 1:2000 dilution of HRP-conjugated anti-IgG (Cell Signaling Technology). After a final TBS-T wash to remove any unbound antibody, a 1:1 ratio mix of ECL Advance Western Blotting Detection kit reagents was then applied to the membrane and left for 1 minute, before visualizing HRP-conjugated proteins using a Bio-Rad Gel Doc XRS+ System. Membranes were then re-stained with an anti-α-Tubulin primary antibody (Cell Signaling Technologies) and the detection process was repeated in order to produce a loading control.