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Fig. 6 | Cell Communication and Signaling

Fig. 6

From: Low levels of pyruvate induced by a positive feedback loop protects cholangiocarcinoma cells from apoptosis

Fig. 6

HDAC3 deacetylates cMYC at K323 and protects cMYC from ubiquitinated degradation. a HuCCT1 cells were transfected with HA-tagged HDAC1–3 plasmid for 36 h, then cells were lysated and immunoprecipitated with HA-beads. The input was visualized by Western blotting with the specific antibody. The immunoprecipitated sample was washed and visualized by Western blotting with the antibody for HA and cMYC. b HuCCT1 cells treated with 100μg/ml CHX (Cycloheximide), with or without 10 μM RGFP966 for 6 h. Cells were lysated and visualized by Western blotting. c HuCCT1 cells treated with 10 μM MG132 with or without 10 μM RGFP966 for 24 h. Cells were lysated and visualized by Western blotting. d HDAC3 deacetylated toward cMYC (K323) acetylated peptides were assayed in the presence of 10 μM RGFP966 and 100 μM pruvate. The deacetylated peptides were analyzed by MS are shown. e and f HuCCT1 cells were transfected with wildtype and mutant flag tagged cMYC plasmid and then treated with 10 μM RGFP966 for 24 h. The immunoprecipitated protein from cell lysates were analyzed via Western blotting and flag are shown as loading controls. g and h Flag-tagged cMYC and HA-tagged ubiquitin were coexpressed in HuCCT1 and HDAC3-knockdown cells, then treated with 10 μM RGFP966 for 24 h with or without 10 μM MG132. Ubiquitination levels of affinity purified Flag-cMYC proteins were detected and visualized by Western blotting. i HuCCT1 cells were transfected with HA tagged HDAC3 plasmid and then endogenous proteins were visualized by Western blotting

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