Normal rabbit immunoglobulin g igg

PGN and the NF-κB inhibitor Bay11-7082 were purchased from Sigma-Aldrich and Calbiochem, respectively. Rabbit anti-IκBα antibody, rabbit antiphospho-IκBα antibody, rabbit NF-κB/p65 antibody, and normal rabbit immunoglobulin G (IgG) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Mouse anti-ACTB antibody and goat anti-DEFB124 antibody were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

Total cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime). The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on either 10% or 15% polyacrylamide gels and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked for 2 hours at room temperature and incubated with primary antibodies. After washing, the membranes were incubated with the appropriate secondary antibodies. The protein bands were visualized using an enhanced chemiluminescence system. Antibodies used in this study included: normal rabbit immunoglobulin G (IgG) [2729], anti-His [2365], and anti-Ubiquitin [3936] from Cell Signaling Technology (Danvers, MA, USA); anti-PSMD14 (ab109130), anti-AGR2 (ab76473), and anti-β-Actin antibodies (ab8227) from Abcam (Cambridge, MA, USA); and anti-HA (H6908) and anti-Myc (06-549) from Sigma-Aldrich (St Louis, MO, USA).