Bax n20

MIN6 cells were lysed in cell lysis buffer containing 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 10 mm dithiothreitol, 1 mm Na₃VO₄, and complete protease inhibitor mixture. Equal amounts of protein were resolved by 10% or 4–12% NuPAGE (Invitrogen) gels, transferred onto PVDF membrane (Millipore), and blots were probed with antibodies against IER3IP1 (sc-84849; Santa Cruz), Puma (7467; Cell Signaling Technologies), Bim (202000; Calbiochem), cleaved caspase-3 (9661; Cell Signaling Technologies), p-FoxO1/Fox3a (9461; Cell signaling), FoxO1 (2880; Cell signaling), Fox3a (2497; Cell signaling), β-actin (A-2066; Sigma), Bax (6A7) (2281-MC-100; Trevigen), Bax (N20) (sc-493, Santa Cruz), Bak (upstate, 06536), p-IRE1α (NB100-2323; Novus), IRE1α (3294; Cell signaling), p-PERK (3179, Cell signaling), PERK (sc-9477; Santa Cruz), ATF4 (ARP37017_P050, Aviva systems biology); p-eIF2a (9721, Cell signaling), eIF2a (9722; Cell signaling), and Chop (2895; Cell signaling). To detect BAX activation, immunoprecipitation (IP) was performed using 1% CHAPS buffer (1% CHAPS, 142.5 mM KCl, 2 mM CaCl₂, 20 mM Tris-Cl, pH 7.4). Anti-6A7 IP was performed using 1% CHAPS buffer. Antibody detection was accomplished using enhanced chemiluminescence (PERKinElmer) and LAS-3000 Imaging system (FUJIFILM).

MIN6 cell culture, RNA isolation, and first-strand cDNA synthesis, and preparation of pLKO.1-Pdx1 shRNA lentivirus were performed as previously described (6 (link)). TaqMan assay numbers were: Hmbs, Mm00660262; Pdx1, Mm00435565; Bax, Mm00432051; and Bak, Mm00432045. The pLKO-Bax shRNA (RMM4533), Bak shRNA (RMM4534) were purchased from Thermo Scientific. Lentivirus was added to the medium on day 1. The blots were probed with antibodies against Pdx1 (07-696; Millipore), cytochrome c (mouse6H2.B4, Millipore), Actin (A-2066; Sigma), Bax (6A7) (2281-MC-100; Trevigen), Bax (N20) (SC493, Santa Cruz Biotechnology) and Bak (06536, Millipore). To detect BAX activation, immunoprecipitation (IP) was performed using 1% CHAPS buffer (1% CHAPS, 142.5 mm KCl, 2 mm CaCl₂, 20 mm Tris-Cl, pH 7.4). Anti-6A7 IP was performed using 1% CHAPS buffer. Antibody detection was accomplished using enhanced chemiluminescence (PerkinElmer) and LAS-3000 Imaging system (FUJIFILM).

The postmortem human brain formalin-fixed paraffin-embedded tissue sections for immunostaining were obtained from BioChain (Newark, CA, USA), Biomax (Derwood, MD, USA), and Rush Alzheimer’s Disease Center (RADC, Chicago, IL, USA). The frozen postmortem human brain tissues for immunoblotting were from BioChain. This study was granted with exemption by the Institutional Review Board (IRB) at the Illinois Institute of Technology (Legacy-IRB-2019-017). Antibodies against total-Tau (T-Tau; D1M9X), phosphorylated Tau (P-Tau; T181), P-Tau (T205), TIA-1, and Bax (D2E11) were purchased from Cell Signaling Technology (Danvers, MA, USA); Bax (N20) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The Bax∆2 monoclonal antibody (2D4), generated against amino acids (GFHGSSRANG) unique to Bax∆2, is well characterized, and was confirmed not to cross-react with Baxα in either immunostaining or immunoblotting [13 (link),32 (link)].

Whole brain lysate from wildtype and knock-out animals was analyzed by Western blot for validation of gene knock-out using the following antibodies: Bax N20 (Santa Cruz, SC-493, 1:1000); Caspase-3 (BD Biosciences, 559565, 1:1000); Sarm1 (Abcam, ab226930, 1:1000); beta-Actin (Sigma, A5316, 1:1000).