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. 2020 May 5;39(23):4551–4566. doi: 10.1038/s41388-020-1312-6

Fig. 7. LRRC4 promoted the degradation of DEPTOR protein, and re-expression of DEPTOR restored autophagy activation.

Fig. 7

a Representative western blotting analysis of LRRC4, DEPTOR, LC3B and GAPDH in U251, PG1 and PG2 cells with or without transfection with LRRC4. Numbers represent the relative ratio of DEPTOR/LC3B and GAPDH. b Western blotting analysis was used to measure of the half-life of DEPTOR after treatment with cycloheximide in U251 cells with (down) or without (upper) LRRC4 transfection. Cells were lysed and protein was extracted at 0, 4, 8, 16, 24 h after cycloheximide treatment. c DEPTOR ubiquitination was assessed by an anti-GFP antibody in the presence of MG132 when GFP-DEPTOR, HA-ubiquitin, and LRRC4 or vector were co-transfected into U251 cells. d Representative western blotting analysis of LRRC4, p-MTOR, MTOR, p-ULK1, ULK1, p-S6K, S6K and Tubulin in U251 and PG2 cells with or without transfection with LRRC4. e Representative western blotting analysis of LRRC4, p-MTOR, p-S6K, S6K, P62, LC3B and GAPDH in U251 cells, U251 cells with or without LRRC4 transfection were treated with dimethylsulfoxide (DMSO) or Rapamycin. f Representative western blotting analysis of LRRC4, DEPTOR, LC3B and GAPDH in U251 cells, U251 cells with stable expression LRRC4 and LRRC4-expressing U251 cells with ectopic expression of DEPTOR. g Autophagosomes were observed by transmission electron microscopy in U251 cells, U251 cells with stable expression LRRC4 and LRRC4-expressing U251 cells with ectopic expression of DEPTOR.