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. 2020 Nov 10;9:e55994. doi: 10.7554/eLife.55994

Figure 5. Increased glutathione drives decreased GAPDH-Rheb binding in S47 cells.

(A) WT and S47 lung (left) and skeletal muscle (center) were assessed for GSH/GSSG ratio (mean ± SD, n = 3). WT and S47 immortalized MEFs (iMEFs), either untreated or treated with 50 μM DEM for 5 hr, were analyzed for GSH/GSSG ratio (mean ± SD, n = 4). (B) WT and S47 iMEFs were untreated or treated with 50 μM of DEM or 50 μM of DEM + 0.5 mM glutamate for 5 hr and protein lysates were analyzed by western blot for indicated mTOR markers. (C) Whole cell lysates were extracted from WT and S47 mouse lung (left) and skeletal (center) tissue. Proteins were cross-linked with BMH, resolved by SDS/PAGE, and detected by western blotting with a GAPDH specific antibody (Top). Untreated protein lysates were analyzed by western blot analysis for total GAPDH (Bottom). WT and S47 iMEFs were treated with 50 μM of DEM for 5 hr and protein lysates were analyzed as described (right). (D) WT cells were treated with PBS or 3 mM GSH for 24 hr. S47 cells were treated with PBS for 24 hr, PBS for 5 hr, 100 μM BSO for 24 hr, or 50 μM DEM for 5 hr. IP of the lysates with anti-Rheb followed by western analysis for associated GAPDH and Rheb (top panel). The same lysates were analyzed by western blotting for GAPDH and Rheb (bottom panel). (E–F) Proximity ligation analysis (PLA) was performed in WT and S47 MEFs treated with 50 μM of DEM for 5 hr and analyzed as described in Figure 4A–B. Scale bar is 30 μm.

Figure 5.

Figure 5—figure supplement 1. Glutathione depletion by BSO alters GAPDH cross-linking and the GAPDH-Rheb interaction.

Figure 5—figure supplement 1.

(A) WT and S47 cells were treated with PBS or 100 μM BSO for 24 hr. Lysates were prepared, treated with or without BMH, run on SDS-PAGE and detected by western blotting with a GAPDH antibody. (B) WT and S47 cells were treated with DMSO or 2 μM Erastin for 24 hr. Lysates were prepared, treated with or without BMH, resolved by SDS-PAGE, and probed by western blotting for GAPDH. (C) An in situ proximity ligation assay was performed in WT and S47 MEFs that are treated with 10 μM of BSO for 24 hr. Shown on the right is the quantification of the mTOR-Rheb interactions, measured as the average number of PLA signals per nuclei. Scale bars represent 30 µm.