Immunoblotting
LYSIS
For the immunoblotting of eIF2α phosphorylation and selective ATF4 translation, cells were treated with 4OH-TAM (10 μM, 15 μM and 20 μM) for 8 h or with 10 μM for 2, 4 and 8 h. Whole cell lysate was extracted from the RIPA lysis solution (Abcam, Cat. No. ab156034) with the Complete Protease Inhibitor Cocktail Tablets (Roche Applied Science, Cat. No. 11697498001) and PhosSTOP phosphatase inhibitor (Roche Applied Science, Cat. No. 4906845001). Equal amounts of protein (50 μg) were boiled for 5 min in 1 × Laemmli sample buffer (BioRad, Cat. No. 1610747) and separated on Mini-PROTEAN precast gels (BioRad, Cat. No. 4569034). The binding of antibody (ERα Antibody (Santa Cruz, Cat. No. sc-8002, clone F-10, RRID: AB_627558), Phospho-eIF2α XP antibody (Ser51) (Cell Signaling Technology, Cat. No. 3398, clone D9G8, RRID: AB_2096481), eIF2α antibody (Cell Signaling Technology, Cat. No. 5324, clone D7D3, RRID: AB_10692650), ATF-4 antibody (Cell Signaling Technology, Cat. No. 11815, clone D4B8, RRID: AB_2616025), Phospho-Stat5 Antibody (Thermo Fisher Scientific, Cat. No. 71-6900, RRID: AB_2533991), Stat5 antibody (BD Biosciences, Cat. No. 610191, RRID: AB_397590), JAK2 antibody (Thermo Fisher Scientific, Cat. No. AHO1352, clone 691R5, RRID: AB_2536334), Phospho-JAK2 (Tyr1007/1008) Antibody (Cell Signaling Technology, Cat. No. 3771, RRID: AB_330403), Phospho-JAK2 (Tyr221) Antibody (Cell Signaling Technology, Cat. No. 3774, RRID: AB_390750), Anti-VDAC1/Porin Antibody (Santa Cruz, Cat. No. sc-390996, clone B-6, RRID: AB_2750920) and α-Tubulin antibody (Sigma-Aldrich, Cat. No. T5168, clone B-5-1-2, RRID:AB_477579)) was detected using an enhanced chemiluminescence horseradish peroxidase (HRP) substrate (Thermo Fisher Scientific, Cat. No. 32106). All primary antibodies were used at a dilution of 1:1000, expect α-Tubulin antibody, which was used at a dilution of 1:5000. Imaging and band quantification were carried out using Odyssey Fc Imaging System and Image J/Fiji Software.