Anti ire1α

Total ovary lysates were separated on SDS-PAGE. After electrophoresis, the protein was transferred to PVDF membrane. The membranes were blocked with 5% skimmed milk and incubated with anti-GRP78 (1:1000, #11587–1-AP, Proteintech, Hubei, China), anti-PARP1 (1:4000, #66520-1-lg, Proteintech), anti-PAR (1:1000, #4335-MC-100, Bio-techne, Minneapolis, USA), anti-mTOR (1:1000, #20657-1-AP, Proteintech), anti-p-mTOR (1:800, #sc-293133, Santa cruz, Dallas, Texas, USA), anti-KitL (1:1000, #bs-0545R, Biorbyt, Hubei, China), anti-p-IRE1α (1:800#, bs-16698R, Biorbyt), anti-IRE1α (1:1000, #27528-1-AP, Proteintech), anti-ATF4 (1:1000, #10835-1-AP, Proteintech), anti-p-PERK (1:800, #Orb336657, Biorbyt), anti-PERK (1:1000, #24390-1-AP, Proteintech), anti-HA-tag (1:1000, #30701ES60, YEASEN), anti-FLAG-tag (1:1000, #30501ES60, YEASEN) and anti-GAPDH (1:4000, #ab8245, Abcam, Cambridge, UK) antibodies overnight at 4 °C. After TBST washing, the membranes were treated with the corresponding peroxidase-conjugated secondary antibody, and then the signal was detected with an Enhanced Chemiluminescence Detection Kit (#32106, Thermo Scientific, Waltham, MA, USA) on Tanon 4500 gel imaging system (Shanghai, China).

Lung tumor tissues and cells were lysed in RIPA lysis buffer and BCA kit was adopted to test the concentration of total protein (E-BC-K318-M, Elabscience, Wuhan, China). Then, protein was separated, and transferred into PVDF membrane. After blocking with skimmed milk, the membranes were incubated overnight anti-IRE1α (27,528-1-AP, 1:2000), anti-XBP1s (24,868-1-AP, 1:6000), anti-GRP78/BIP (GRP78/BIP, 1:10,000), anti-CHOP (15,204-1-AP, 1:3000), anti-PERK (24,390-1-AP, 1:2000), and anti-β-actin (81,115-1-RR, 1:20,000) (Proteintech, Wuhan, China); anti-E-cadherin (#3195, 1:1000), and anti-N-cadherin (#13,116, 1:1000) (CST, Boston, MA, USA); and Vimentin (sc-373717, 1:5000, Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA). Second antibody (anti-Rabbit IgG-HRP, SA00001-2, 1:10,000, Proteintech, Wuhan, China) was subsequently incubated for 2 h. Finally, immunoblots were performed by the enhanced chemiluminescence (ECL) kit (34,577, Thermo Fisher Scientific, Waltham, MA, USA), and visualized by Tanon 5200 (Tanon, Shanghai, China).

Cell or tissue lysates and prestained protein molecular weight markers (#26612, Pierce, USA) were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), followed by transferring onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in Tris‐buffered saline with 0.5% Tween 20 (TBST) containing 5% bovine serum albumin (BSA) and probed with primary antibodies overnight at 4℃. Then, the membranes were washed three times and incubated with the horseradish peroxidase (HRP)‐conjugated secondary antibodies goat‐anti‐rabbit IgG (#7074) and horse‐anti‐mouse IgG (#7076). The primary antibodies used in the study were anti‐p300 (#86377), anti‐p‐IRE1α (#PA1‐16927, Invitrogen, USA), anti‐IRE1α (#3294), anti‐XBP1s (#24868‐1‐AP, Proteintech, USA), anti‐acetylated lysine (Ac‐K; #9681), anti‐activating transcription factor 4 (ATF4; #11815), anti‐glucose regulated protein 94 (GRP94; #2104), anti‐binding immunoglobulin protein (BIP; #3183), anti‐C/EBP homologous protein (CHOP; #2895), anti‐ubiquitin (#3933), anti‐Herpud1 (ab150424, Abcam, USA), and anti‐GAPDH (#2118). Antibodies whose supplier is not stated were purchased from Cell Signalling Technology (USA). Immunoreactivity was visualized by enhanced chemiluminescence (ECL, #34577, Pierce, USA).