Methylglyoxal

Tissues were fixed in 4% formaldehyde and embedded in paraffin before 2 µm slices were cut for hematoxylin-eosin staining (Roth, Fluka). Protein isolation and immunoblotting from quadriceps muscle was performed as previously described^{32 (link)}. If not stated differently, immunoblots were cropped from the same gel. The following antibodies were used for immunoblots: αTubulin (T6074, Sigma Aldrich), phospho-AMPK^Thr172 (#2531, Cell Signaling), total AMPK (#2603, Cell Signaling), BIP (#3183, Cell Signaling), phospho-eIF2α^Ser51 (#3597, Cell Signaling), total eIF2α (#5324, Cell Signaling), GPX1 (AF3798-SP, R&D Systems), GPX4 (ab125066, Abcam), IRE1α (#3294, Cell Signaling), Methylglyoxal (STA-011, Cell Biolabs), MFN2 (#9482, Cell Signaling), OXPHOS (#MS60, MitoSciences/Abcam), PGC1α (ab71130, Abcam), PHGDH (14719-1-AP, Proteintech).

Whole cell lysates were prepared as described for quantitative proteome comparison. Cell lysates were centrifuged at 15000 g for 2 min, and protein concentration in the supernatant was determined with a bicinchoninic acid protein assay (BCA, Pierce). Equal amounts of protein extracts (5 to 40 μg depending on the antibody used) were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes using a semi-dry blotting system (Bio-Rad, Hercules, CA, USA). For immune detection of proteins, membranes were probed with the following antibodies: DJ-1 (R&D Systems 3668, Minneapolis, MN, USA), GAP43 (Böhringer 1379011, Ingelheim, Germany), β3-Tubulin (Sigma T8660, Kavasaki, Japan), Cathepsin B (R&D Systems AF953), methylglyoxal (MGO, Cell Biolabs Inc. STA-011, San Diego, CA, USA). β-Tubulin (Sigma Aldrich T6199) or GAPDH (abcam 9484) were used as loading control. methylglyoxal (MGO) immunoblots were stripped by incubating the membrane with pre-heated stripping buffer (2% SDS, 62.5 mM Tris-HCl pH 6.8, 1:125 v/v ß-mercaptoethanol) at 50 °C for 45 min, followed by rinsing for 1–2 h with water and 5 min with TBST before they were blocked and re-probed with GAPDH antibody. Signal intensity was analyzed with ImageJ [38 (link)].