AIP1 is critical in transducing IRE1-mediated endoplasmic reticulum stress response - PubMed (original) (raw)

ER stress-induced activation of ASK1-JNK and apoptosis are blunted in AIP1-KO cells. Mouse lung microvascular EC (MLEC) and MEFs were isolated from AIP1-WT and AIP1-KO. a, MLEC were treated with TNF (10 ng/ml), thapsigargin (Tsg, 5 μ

m

), or tunicamycin (Tm, 1 μ

m

) for the indicated times. ASK1 activity was determined by an in vitro kinase assay using GST-MKK4 as a substrate. Activation of JNK was detected by Western blotting with a phospho-specific antibody against p-JNK1/2. Total JNK was determined by Western blot with anti-JNK1. b, MLEC were treated with oxidative stress stimuli H2O2 (1 m

m

), buthionine-sulfoximine (BSO, 0.5 m

m

), or paraquat (1 m

m

). Activation of JNK was detected as described in a. c, MEFs were treated with ER stress and oxidative stress as indicated for 30 min. Activation of JNK was detected as described in a. AIP1 and β-tubulin proteins were determined by Western blot with respective antibodies. d and_e_, MLEC were treated with Tm (1 μ

m

) or TNF (10 ng/ml) plus cycloheximide (CHX, 10 μg/ml) for 6 h. EC apoptosis was also determined with annexin-V/PI staining followed by FACS analysis. Representative FACS is shown in d, and the % of apoptotic cells (both upper and lower right quadruplets in FACS) are quantified in e.f, MEFs were treated with Tm (1 μ

m

) or TNF (10 ng/ml) plus cycloheximide (CHX, 10 μg/ml) for 6 h. EC apoptosis was determined as in d and e. Data are presented in e and f as mean of duplicates from two independent experiments. * indicates a statistical significance between WT and AIP1-KO cells; p < 0.05. Error bars show the calculated S.D.