Anti gsh antibody

For immunoblotting analysis, cells were lysed in RIPA buffer supplemented with protease inhibitors cocktail (Roche). Protein concentration was determined using the detergent-compatible protein assay (Bio-Rad). Cell lysates containing equal amount of protein were subjected to SDS-PAGE. Proteins were then transferred to a polyvinylidene difluoride membrane (PVDF), and probed with appropriate primary antibodies overnight at 4 °C. The following antibodies were used in immunoblot analysis: TM9SF4 antibodies (1:2000, Proteintech), β-actin antibodies (1:2000, Santa Cruz), ERK antibodies (1:2000, Cell Signaling Technology), β-tubulin antibodies (1:1000, Santa Cruz), V5 antibodies (1:2000, Thermo Fisher), GFP antibodies (1:2000, TransGen Biotech), anti-GSH antibody (1:1000, Thermo Fisher) and cofilin antibodies (1:3000, Proteintech). Then, the membrane was incubated with HRP-conjugated secondary antibody (1:5000, Cell Signaling Technology) for 2 h at room temperature, followed by detection using ECL substrate (GE Healthcare). The images of protein bands were analyzed in a semi-quantitative manner through ImageJ 1.52p software. The uncropped scans of the blots were provided in the Source Data file.

Glutathionylated proteins were detected by Western blot analysis of cellular lysates after nonreducing SDS-PAGE. Cells were collected by trypsinization and centrifugation at 500 ×g and then resuspended in PBS, pH 7.4, containing the protease inhibitor PMSF and supplemented with 5 mM N-ethylmaleimide (NEM) to block unreacted thiol group. Total cellular proteins (50 μg per lane) were separated on 12% (w/v) SDS-PAGE and transferred to nitrocellulose membranes. Glutathionylated proteins were visualized with anti-GSH antibody (1 : 1000, Thermo Fisher Scientific number MA1-7620). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma) was used as loading control. After several washes in Tween/Tris-buffered saline solution (TTBS), the membrane was incubated for 60 minutes with an anti-rabbit or anti-mouse IgG peroxidase-conjugate antibody (diluted 1 : 5000). Immunodetection was then performed with the enhanced chemiluminescence (ECL) (Bio-Rad, Milan, Italy). The VersaDoc imaging system was used to perform densitometric analysis (Bio-Rad, Milan, Italy).

Cells grown on coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, followed by incubation with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-GM130 antibody (1:1000, Proteintech), anti-GSH antibody (1:1000, Thermo Fisher), anti-paxillin antibody (1:500, Proteintech) and anti-ezrin antibody (1:250, NOVUS Bilogicals). The cells were then incubated with secondary antibodies (Alexa Fluor 488 donkey anti-mouse IgG/ Alexa Fluor 555 donkey anti-mouse IgG, 1:5000, Thermo Fisher) for 1 h at room temperature. DAPI (1:1000, Thermo Fisher) was used for nuclear staining. For F-actin staining, cells were fixed in 4% paraformaldehyde, blocked for 1 h and subjected to FITC-conjugated/rhodamine-conjugated phalloidin (1:1000, Thermo Fisher) staining at room temperature. The percentage of cells containing thick actin stress fiber with a diameter >0.5 μm was analyzed and used as an index for coarse quantification of the stress fiber. Immunofluorescence signal was detected using the Olympus FV1200 confocal system (FLUOVIEW Ver.4.2 software).