Anti p bad

Briefly, equal amounts of proteins were separated from a polyacrylamide gel and electro‐transferred to polyvinylidene fluoride membranes (Millipore Corp.). Membranes were blocked overnight with the primary antibodies. After incubating with the secondary antibodies, the membranes were washed five times with TBST (pH 7.6) for 30 min at room temperature. Chemiluminescence reagent SuperSignal™ West Femto Substrate (Thermo Fisher Scientific) was used to develop the membranes according to the manufacturer's instruction. Immunoreactive bands were visualized using the BIO‐RAD ChemiDoc XRS Imaging System according to the manufacturer's instruction. Densitometric analysis was performed with Quantity One software (Bio‐Rad). The antibodies used were anti‐TNFa (Abcam, #ab66579), anti‐FAS (Abcam, #ab82419), anti‐Bax (Proteintech, #50599‐2‐Ig), anti‐Bcl‐2 (Abcam, #ab196495), anti‐cleaved caspase‐3 (Abcam, #ab49822), anti‐IL6 (Abcam, #ab208113), anti‐Akt (Cell Signaling Technology, #4685), anti‐p‐Akt (Cell Signaling Technology, #4060), anti‐beta‐actin (Proteintech, 60008‐1‐Ig), anti‐P‐BAD (Abcam, ab129192), anti‐p‐Bax (Abcam, ab111391), and anti‐p‐Par4 (Cell Signaling Technology, #2329).

LPS (Escherichia coli serotype) and PIM-447 were purchased from Sigma (St. Louis, Mo, USA) and Selleck Chemicals (Houston, TX, USA), respectively. Recombinant PIM-1, PIM-2, and PIM-3 kinases were purchased from Life Technologies (Carlsbad, CA, USA), Abcam (Cambridge, UK), and Millipore (Burlington, MA, USA), respectively. The IMAP™ FP Screening Express Kit was obtained from Molecular Devices (San Jose, CA, USA). The 5-FAM-labelled Bad peptide (5-FAM-RSRHSSYPAGT) was purchased from AnaSpec (Fremont, CA, USA). Dithiothreitol was obtained from Sigma. The primary antibodies against TLR4, p-IKK α/β, p-NFκB/P65, iNOS, MyD88, ASC, caspase-1, and NLRP3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-IL-1β antibody was purchased from Novus Biologicals (Centennial, CO, USA). Anti-p-Bad and anti-PIM-1 antibodies were purchased from Abcam (Cambridge, UK). Anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louise, MO, USA). Anti-horse anti-mouse IgG-horseradish peroxidase (HRP) and anti-goat anti-rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PIM-1 siRNA was purchased from Life Technologies (Carlsbad, CA, USA).