Figure 4.

SCF^Fbxo22 ubiquitinates KDM4B complexed with NCOR1 for degradation, which is critical for induction of TFEB transcription and an acquired stress resistance. (A) Lysates from RPE-1 cells expressing the indicated Dox-inducible shRNA and/or WT KDM4B or its mutants after (24 h) or before (0 h) preconditioning with serine depletion, were subjected to immunoblotting using the indicated antibodies. (B) Cells treated as in (A) were then treated with low glucose (2 mM) media for 48 h. The proportions of dead cells (upper panel) and the relative cell numbers (lower panel) were determined at the indicated times as in Figure 1A. (C) Lysates from RPE-1 cells expressing the indicated Dox-inducible shRNA after (24 h) or before (0 h) preconditioning with serine depletion were subjected to immunoblotting using the indicated antibodies. (D) Cells treated as in (C) were then treated with low glucose (2 mM) media for 48 h. The proportions of dead cells (upper panel) and the relative cell numbers (lower panel) were determined at the indicated times as in (A). (E) Lysates from HeLa cells expressing the indicated genes were sequentially immunoprecipitated with anti-FLAG M2 affinity gel and anti-KDM4B antibodies. The resultant precipitates were subjected to immunoblotting using the indicated antibodies. (F) Lysates from HeLa cells expressing WT FBXO22 or its deletion mutants were immunoprecipitated with an anti-FLAG M2 affinity gel and then subjected to immunoblotting using the indicated antibodies. ΔFC: FIST-C domain deletion, ΔFN: FIST-N deletion (G) Lysates from RPE-1 cells expressing the indicated genes were subjected to immunoblotting using the indicated antibodies. (H) Lysates from HeLa cells expressing the Dox-inducible shControl (C) or shFBXO22 (F) were immunoprecipitated and then subjected to immunoblotting using the indicated antibodies. (I) Lysates from HeLa cells expressing FLAG-HA-KDM4B (FH-KDM4B) with the Dox-inducible shControl or shFBXO22 were sequentially immunoprecipitated using anti-FLAG M2 affinity gel and anti-HA affinity gel. The resultant immunoprecipitates were subjected to immunoblotting. (J) WT (W) or FBXO22^−/− (K) HeLa cells were transfected with the HA-Ubiquitin (HA-Ub), treated with or without MG132, lysed under denaturing conditions, and subjected to immunoprecipitation with the indicated antibodies, followed by immunoblotting. (K) FBXO22^−/− HeLa cells were transfected with the indicated genes, treated with MG132, lysed under denaturing conditions, and subjected to StrepTactin pulldown, followed by immunoblotting. Data are presented as means±s.d. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons post hoc test was performed against a control (shCont, WT, or C) group mean (B, D). ***P < 0.001, ****P < 0.0001