Anti γ gcs

Except for the lactate dehydrogenase activity kit (Labtest, Lagoa Santa, MG, Brazil), SYBR Safe DNA gel stain (Life Technologies, Carlsbad, CA, USA), and anti-γ-GCS, HO-1, and NQO-1 primary antibodies (Santa Cruz Biotechnology Inc., Dallas, TX, USA), all other chemicals were purchased from Sigma-Aldrich (São Paulo, SP, Brazil).

Hispidin (purity > 99%) was synthesized as previously reported [34 ]. RPMI 1640, DMEM, FBS and 0.25% trypsin were purchased from Thermo Fisher Scientific (Shanghai, China). MTT, necrostatin-1, hemoglobin, N-acetyl-L-cysteine (NAC), Catalase, Carboxy-PITO, the TUNEL kit, Hoechst 33342, rapamycin, 3-Methyladenine, wortmannin, bafilomycin A1, hydroxychloroquine, E64d and pepstatin A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cathepsin inhibitor 1 was purchased from AbMole BioScience (Shanghai, China). The broad-spectrum caspase inhibitor z-VAD-fmk was purchased from BD Biosciences (San Jose, CA, USA). The mCherry-GFP-LC3B reporter was purchased from Beyotime Biotechnology (Jiangsu, China). The mouse anti-caspase-8 monoclonal antibody, rabbit monoclonal anti-caspase-3, anti-β-actin, anti-caspase-9, anti-LC3A/B and anti-STMN1 phosphorylated ser¹⁶ antibodies were from Cell Signaling Technology (Beverly, MA, USA). The anti-γ-GCS, horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). STMN1 siRNA SMART pools were purchased from Dharmacon (Lafayette, CO, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). A tubulin polymerization assay kit was purchased from Cytoskeleton (Denver, CO, USA)

Cells were harvested in ice-cold PBS and lysed in RIPA buffer (Cell signaling) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche) for 20 min on ice. Fifty millimolar of N-Ethylmaleimide (NEM; Sigma) was supplemented for s-glutathionylation preparations. After centrifugation at 15,000×g for 15 min at 4 °C, total protein in whole-cell extracts was quantified using Rotiquant (Carl Roth). Forty micrograms of protein were resolved by SDS-PAGE (Invitrogen) and blotted on PVDF membranes (Invitrogen). The membranes were probed with anti-GSTP1, anti-xCT, anti-catalase, anti-SOD1, anti-GPX1, anti-γGCS, or anti-β actin (Santa Cruz) primary antibodies followed by secondary horse-radish peroxidase (HRP) coupled antibodies (Santa Cruz). Signals were acquired in a chemiluminescence detection system (Applied Biosystems) in a linear dynamic range.