Anti fth1

Purified erianin (>98%) (Cat: B20844) was gained from Shanghai Yuanye Biological Co., Ltd. All antibody follow-up experiments used were anti-xCT (Cat: ab175186), anti-Glutaminase (Cat: ab93424), anti-GPX4 (Cat: ab125066), anti-Heme Oxygenase-1 (Cat: ab189491) were from Abcam (Cambridge, United Kingdom), anti-FTH1 (Cat: 4393S), anti-NRF2 (Cat:12721S), anti-GAPDH (Cat: 5174S) were provided by Cell Signaling Technology (Danvers, United States). And inhibitors mentioned in experiments were deferoxamine (Cat: S5742) (Selleck, Houston, United States), chloroquine (Cat: C6628) (Sigma-Aldrich, St. Louis, United States), Z-VAD-FMK (Cat: HY-16658B), N-Acetylcysteine (Cat: HY-B0215), necrostatin-1 (Cat: HY-15760), L-Glutathione reduced (Cat: HY-D0187), TBHQ (Cat:HY-100489) were obtained from MedChem Express (New Jersey, United States), pLVX-U6-NRF2-shRNA1-PGK-EGFP-E2A-Puro (Cat:p24452) (miaolingbio, Wuhan, China).

Cultured cells were harvested and lysed in RIPA buffer containing 1% protease inhibitor. Western blot assays were performed as previously described^{44 (link)}. The following antibodies were used: anti-BRD4 (ab75898, 1:1000) and anti-GPX4 (ab125066, 1:1000) were purchased from Abcam (Cambridge, MA, USA); anti-G9a (#3306, 1:1000), anti-SIRT1 (#9475, 1:1000), anti-LAMP1 (#9091, 1:1000), anti-S6 kinase (#2708, 1:1000), anti-LC3B (#3868, 1:1000), anti-SQSTM1/p62 (#8205, 1:1000), anti-ATG5 (#12994, 1:1000), anti-ATG7 (#8558, 1:1000), and anti-FTH1 (#4393, 1:1000) were purchased from Cell Signaling Technology; and anti-β-tubulin, used as the internal control, was purchased from Santa Cruz Biotechnology (TX, USA, CAS: KM9002T, 1:1000). After incubation with primary antibodies, membranes were incubated with goat anti-mouse IgG H&L (horseradish peroxidase (HRP)-conjugated) or goat anti-rabbit IgG H&L (HRP-conjugated) for 1.5 h at room temperature. Immunoreactions were then detected with a FluorChem HD2 imager (Protein Simple, USA).

The normal MOVAS cells or MOVAS cells with PPARγ-knockdown were treated with Ang II+Bleo for 5 days and were lysed in NP-40 buffer with protease inhibitor cocktail and the crude lysates were cleared of insoluble debris by centrifugation at 12,000 × g. The lysates were immunoprecipitated with normal IgG or primary antibodies, including anti-FTH1 (#4393, Cell Signaling Technology), anti-HA-tag (#ab9110, Abcam) and anti-Flag-tag (#ab205606, Abcam) in a rotator (Scilogex, Rocky Hill, CT) at 4 °C overnight. The 20 μl protein A/G PLUS-Agarose beads (#sc-2003, Santa Cruz Biotechnology) were added into the 200 μl homogenates or lysates and incubated for 4 h with gentle agitation. The beads were washed 3 times with the lysis buffer and boiled with 10 μl loading buffer. The beads were removed by centrifugation (5 min at 12,000 × g). The supernatant fraction was collected and used for immunoblotting.

The following primary antibodies were used for immunoblot: anti-FTH1 (cat no., 3998S), anti-IRP1(cat no., 20272S), anti-STAT1(cat no., 14994) and anti-pSTAT1(Tyr701) (cat no., 9167) were all purchased from Cell Signaling Technology (Danvers, MA, USA); anti-IRP2 (cat no., NB100-1798), and anti-FPN (cat no., NBP1-21502) were from Novus Biologicals (Centennial, CO, USA); anti-iNOS (cat no., ab178945) were from abcam (Cambridge, MA, USA); anti-Arg1 (cat no., 16001-1-AP) were from Proteintech (Wuhan, China).

Renal cortex or BUMPT cells were homogenized, and the homogenates were centrifuged for the collection of the supernatants. The protein concentration was determined by BCA assay (Beyotime, catalog P0012S). The samples with equal amounts of protein were then subjected to SDS-PAGE analysis and the fractionated proteins were transferred to PVDF membranes (0.22 μm). The membranes were immersed in 5% milk for 1 h to block the background. They were incubated with diluted primary antibodies overnight at 4 °C. The primary antibody used included: anti-NCOA4 (Bethyl Laboratories, catalog # A302-272A; 1:1000), anti-FTH1 (Cell Signaling Technology, catalog # 3998S; 1:1000), anti-GPX4 (Affinity Biosciences, catalog DF6701; 1:1000), anti–NOX4 (Abcam, catalog # ab133303; 1:2000), anti–CHIP (Abcam, catalog # ab134064; 1:2000), anti-β-actin (proteintech, catalog # 66009-1-Ig; 1:5000) and anti-GAPDH (proteintech, catalog # 60004-1-Ig, 1:1000). The membranes were washed with TBST and incubated with secondary diluted antibodies (1:1000) for 1–2 h. Finally, the membranes were washed and subjected to evaluation by ECL chemiluminescence detector.

Whole cell extracts and western blotting were performed as previously described [37 (link)]. Antibodies against NCOA4 was from Bethyl, anti-FTH1, anti-LC3 were from Cell Signaling. Anti-CD71 antibody was purchased from Proteintech. anti-β-ACTIN was from Sigma-Aldrich.