Fig. 4

Hdac3 and NCoR1 repressors degrade by autophagy. a NCoR1 and Hdac3 co-localize with Lamp2 protein in primary hepatocytes. Primary hepatocytes treated for 24 h with 100 nM BafA1 were PFA fixed and co-stained with primary antibodies, secondary anti-rabbit IgG Alexa Fluor 568 and anti-rat IgG Alexa Fluor 488 antibodies were used for detection. The white arrowheads indicate double-positive cytosolic structures. Scale bar: 40 µm. b Immunoblot analyses of GFP-Trap agarose eluates with indicated antibodies. HEK293T cells were transfected with GFP-GABARAP, GFP-LC3 or GFP expressing plasmid constructs. Twenty-four hours post-transfection cells were treated with Vps34 inhibitor PIK-III (2.5 μM) for 6 h before collection and proceeding with immunoprecipitation using GFP-Trap agarose. c Immunoblot analyses of GFP-Trap agarose eluates with indicated antibodies. HEK293T cells were transfected with GFP and GFP-GABARAP with or without Vps15-Flag expressing plasmid constructs. Twenty-four hours post-transfection cells were collected for immunoprecipitation using GFP-Trap agarose. d Immunoblot analyses of PPARα-containing complexes immunoprecipitated from HEK293T cells. HEK293T cells were transiently transfected with indicated plasmid constructs. Cells were collected 24 h post-transfection and PPARα complexes were immunoprecipitated using anti-HA antibody. The quantification of endogenous Hdac3 protein in PPARα immunoprecipitates normalized to the non-specific binding to beads is presented as fold change difference over empty vector-transfected condition. Data are means ± SEM (n = 8, P < 0.05 *: vs empty vector-transfected cells). e Proximity ligation assay between endogenous Hdac3 and ectopic PPARα-HA protein in HEK293T cells co-transfected with empty vector or Vps15-Flag expressing plasmid constructs (in all conditions GFP-expressing plasmid was co-transfected). PLA-detected PPARα/Hdac3 interactions (red dots) were quantified in n = 100–150 GFP-positive cells captured on n = 19–21 individual fields. The graph represents the number of PLA interactions as folds over empty vector-transfected condition. Data are means ± SEM (P < 0.05 *: vs empty vector-transfected cells). Scale bar: 10 µm