Ab185544

HT-22 cells were planted on poly-L-lysine coated glass coverslips and cultured in the Hypoxia Incubator Chamber. After OGD treatment, HT-22 cells were permeabilized with 0.3% Triton X-100 for 10 min at room temperature and then washed with PBS for 3 times. In order to reduce nonspecific staining, HT-22 cells were blocked with 3% BSA for 1 h. After washing, the cells were incubated with the primary antibodies, including rabbit anti-LC3 (1 : 200, #4108, Cell Signaling Technology), rabbit anti-SOD2 (1 : 150, NB100-1992, NOVUS), rabbit anti-Trx2 (1 : 200, ab185544, Abcam), and rabbit anti-Prx3 (1 : 100, A3076, ABclonal), overnight at 4°C. On the second day, the primary antibodies were removed and washed 5 times with PBS. HT-22 cells were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1 : 500; A-21206; Thermo Fisher Scientific) for 1 h at room temperature in the dark. After washing, the cell nucleus was stained with DAPI (ab104139, Abcam). Finally, the images were captured with a fluorescent microscope and analyzed with ImageJ software.

The cells were treated under the indicated conditions and then efficient lysed with RIPA buffer at pH 8.0 (150 mM NaCl, 50 mM Tris, 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% SDS). All the samples were quantified using Pierce^TM bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Inc.) to ensure equal loading of proteins. The cellular protein samples were separated by SDS‐PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore). After blocked by 5% BSA, the membrane was probed with the primary antibody and then incubated with HRP‐conjugated secondary antibody in TBST. The primary antibodies include anti‐TXN2 (1:1000 dilution, Abcam, ab185544) and anti‐HP (1:1000 dilution, Abcam, ab256454).

Tissue or cells were lysed in 2x Laemmli buffer and boiled for 5 min at 100 °C followed by centrifugation at 20,000xg for 15 min at 4 °C. The protein extracts were subjected to standard Western blot analysis which was performed. The following antibodies were used for western blotting: Antibody against COX10 (ab84053), TRX2 (ab185544) from Abcam; p-Akt (rabbit, 9271), Akt (rabbit, 9272), p-SMAD1/5/9 (13820), p-SMAD2/3 (8828), SMAD2/3 (8685), SMAD1 (9743), SMAD2 (5339), TFAM (8076) were from Cell Signaling Technology; TGFβR1 (E-AB-70130; recognizing human and mouse), and TGFβR2 (E-AB-60379) were from Elabscience; ALK1 was from Origen (AP01172PU-N) and ALK5 was from R&D (AF3025-sp; recognizing human); β-actin (mouse, A1978) were from Sigma; p-SMAD2 (44–244 G) and SMAD1 (38-5400) were from ThermoFisher. All primary antibodies were diluted 1:1000. For data presented in the same figure panel, the samples were derived from the same experiment and that gels/blots were processed in parallel. Uncropped gel images were provided in Supplementary Fig. 12.