Phospho iκbα ser32 36 5a5

After 24 h post-transfection, cells were collected, and protein extracts obtained by adding Leammli sample buffer (Bio Rad, Ca, USA). Equal amounts of proteins were loaded and separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked using 7.5% milk in TBS/0.01% Tween buffer and then incubated with the indicated antibodies. Primary antibodies were prepared in TBS/0.01% Tween buffer as follows: anti-GAPDH (1:1000) (32233; Santa Cruz Biotechnology, Santa Cruz, California), anti-HA (1:1000) (C29F4; Cell Signaling Technology Europe, B.V), anti-p52NFκB (1:1000) (298; Santa Cruz Biotechnology, Santa Cruz, California), α-Actinin (1:1000) (17829; Santa Cruz Biotechnology, Santa Cruz, California) and Phospho-IκBα (Ser32/36)(5A5) (9246; Cell signaling Technology Europe, B.V) Membranes were washed three times with TBS/0.01% Tween buffer and incubated with HRP coupled secondary anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, California). Finally, Proteins were visualized using the ChemoLuminiscent Reagent (Merck Millipore, Burlington, Massachusetts, U.S) according to the manufacturer’s instructions.

Primary antibodies reactive to β-actin (AC-15; Sigma–Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma–Aldrich, St. Louis, MO, USA), ICAM-1 (15.2; Leinco Technologies, St. Louis, MO, USA), FLIP (Dave-2; Alexis^® Biochemicals, San Diego, CA, USA), IκBα (25; BD Biosciences, San Jose, CA, USA), Mcl-1 (D35A5; Cell Signaling Technology, Danvers, MA, USA), NF-κB p50 (H-119; Santa Cruz Biotechnology, Dallas, TX, USA), NF-κB p65 (C-20; Santa Cruz Biotechnology, Dallas, TX, USA), Poly(ADP-ribose) polymerase (PARP) (C2-10; Trevigen, Gaithersburg, MD, USA), phospho-IκBα (Ser32/36) (5A5; Cell Signaling Technology, Danvers, MA, USA), and phospho-NF-κB p65 (Ser536) (93H1; Cell Signaling Technology, Danvers, MA, USA) were obtained.