Phospho p65 rela ser536

Human thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Tunicamycin was purchased from Sigma. Polyclonal antibodies to ICAM-1, VCAM-1, RelA/p65, β-actin, IκBα, and Lamin B were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to phospho-eIF2α, eIF2α, BiP/GRP78, ATF-4, phospho-AKT, phospho-IKKβ, IKKβ, phospho-(Ser⁵³⁶)-RelA/p65, were obtained from Cell Signaling (Beverly, MA). Tata Binding Protein (TBP) antibody was purchased from Abcam. RelA/p65 transcription factor assay kit was purchased from Cayman Chemical (Ann Arbor, MI) and plasmid maxi kit was from QIAGEN Inc. (Valencia, CA). Protein assay kit and nitrocellulose membrane were from Bio-Rad. Alexa Fluor 488-phalloidin was purchased from Invitrogen. Expression vector encoding Wild type BiP and dominant negative BiP were from Addgene. All other materials were from VWR Scientific Products Corporation (Gaithersburg, MD) and Fisher Scientific (Pittsburgh, PA). SubAB and its non-toxic derivative SubA_A272B were purified as previously described [37] (link).

Thirty micrograms of the protein lysate was resolved by SDS/PAGE (8–10% gel) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Upon the transfer completion, the membranes were probed with antibodies against IκBα, phospho-IκBα (Ser32/36), p65/RelA, and phospho-p65/RelA (Ser536) (Cell Signaling Technology). The bands were visualized with Advanced ECL^® Western Blotting Detection Reagents (Amersham Biosciences, Amersham, UK). Equal protein loading was checked using an anti-GAPDH antibody (Santa Cruz Biotechnology).