Anti-IRE1/ERN1 Antibody Picoband®

Western blot analysis of IRE1 using anti-IRE1 antibody (A00683-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human A549 whole cell lysates,
Lane 2: human SK-OV-3 whole cell lysates,
Lane 3: human PANC-1 whole cell lysates,
Lane 4: rat brain tissue lysates,
Lane 5: mouse brain tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-IRE1 antigen affinity purified polyclonal antibody (Catalog # A00683-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for IRE1 at approximately 170KD. The expected band size for IRE1 is at 110KD.

BCAT1 regulates autophagy during hypoxia by activating ERs via the IRE1-XBP1-RIDD axis. a Western blot analysis of BECN1 and Atg5 in PASMCs cotransfected with BCAT1 and IRE1 siRNA ( n = 5). b Autophagic flux was monitored in PASMCs cotransfected with eGFP-mRFP-LC3 plasmid and control siRNA or IRE1 siRNA that were then exposed to HYP for 24 h. Scale bar = 50 μm ( n = 3). c , d RT-PCR analysis of the mRNA levels of XBP1-s, sparc, pmp2, and Scara3 with rat β-actin serving as the standard ( n = 5). e The formation of autophagosomes was detected, and autophagic activity was estimated in cells in which the expression of XBP1 was knocked down with XBP1 siRNA under HYP for 24 h. Scale bar = 50 µm ( n = 5). Nor normoxia, Hyp hypoxia, H + G hypoxia plus gabapentin, H + NC hypoxia plus control siRNA, H + SI hypoxia plus BCAT1 siRNA, H + SI-IRE1 hypoxia plus IRE1 siRNA, H + SI-XBP1 hypoxia plus XBP1 siRNA, H + Con hypoxia plus control vector, H + B hypoxia plus BCAT1 plasmid, H + Con+NC hypoxia plus control vector plus control siRNA, H + B + Si-IRE hypoxia plus BCAT1 plasmid plus IRE1 siRNA. Statistical analysis was performed with one-way ANOVA. All values are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.
Index in PubMed under a CC BY license. PMID: 32938905

BCAT1 regulates autophagy through the endoplasmic reticulum stress pathway. a Expression of BCAT1 and ER-Tracker Red staining in PASMCs exposed to NOR or HYP for 24 h. Scale bar = 50 μm ( n = 3). b Western blot analysis of PERK, IRE1, ATF6, and GRP78 protein expression in the ERs pathway in PASMCs treated with gabapentin ( n = 8). c Western blot analysis of IRE1, PERK, ATF6, and GRP78 expression in PASMCs transfected with BCAT1 siRNA ( n = 8). d Western blot analysis of BECN1 and Atg5 in PASMCs treated with the ERs pathway inhibitor 4-PBA and BCAT1 plasmid ( n = 8). e Coimmunoprecipitation of the whole-cell lysates of PASMCs exposed to normoxia or hypoxia for 24 h with anti-IRE1, followed by probing with anti-BCAT1 ( n = 3). Nor normoxia, Hyp hypoxia, H + G hypoxia plus gabapentin, H + NC hypoxia plus control siRNA, H + SI hypoxia plus BCAT1 siRNA, N + Con normoxia plus control vector, H + Con hypoxia plus control vector, H + B hypoxia plus BCAT1 plasmid, H + Con+4 hypoxia plus control vector plus 4-phenylbutyric acid, H + B + 4 hypoxia plus BCAT1 plasmid plus 4-phenylbutyric acid, IP immunoprecipitation, IB immunoblotting. Statistical analysis was performed with one-way ANOVA. All values are presented as the mean ± SEM. ** p < 0.01; *** p < 0.001.
Index in PubMed under a CC BY license. PMID: 32938905