Anti nfκb p65 sc 372

Western blot analysis was conducted to determine the level of kinase activation. Cell lysates were collected. One hour after the treatment of HaCaT cells with antidepressants (Flu 0.1 and 0.5 µM; Des 1 and 5 µM; Imi 1 µM) and stimulants (LPS: 3 µg/ml; TNF-α/IFN-γ: 10 ng/ml; DNFB: 1 µM), samples containing an equal amount of protein were separated by SDS–PAGE (4–20% gel; Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Trans-Blot Turbo; Bio-Rad, Hercules, CA, USA). After the transfer, the membranes were cut to allow simultaneous (overnight at 4 °C) incubation with different primary antibodies (anti-NFκB p65 (sc-372), anti-phospho-NFκB p65 (sc-33020), anti-IκB-α (sc-1643), anti-phospho-IκB-α (sc-8404), anti-NIK (sc-7211), anti-phospho-NIK (sc-12957), anti-p38 (sc-7972), and anti-phospho-p38 (sc-101759)) obtained from Santa Cruz Biotechnology (USA) and anti-GAPDH (MAB374, Millipore, USA). The next day, the membranes were washed four times with TBS containing 0.1% Tween-20 (TBST) and then incubated with appropriate secondary antibodies (Vector Laboratories, UK) for 1 h at room temperature. The immunoblots were visualized with a chemiluminescence detection kit (Roche, Germany). The data obtained were normalized to the level of reference proteins and then averaged and presented as a percentage of control ± SEM, from at least three independent experiments.