Fig. 5.
PU.1 protein is recruited by p62 and LC3-II and targeted for selective autophagy degradation in TH9 cells. a The expressions of p62 and PU.1 were analysed by western blot at different time points during TH9 cell differentiation. The western blot shown is representative of two independent experiments. b Quantification of the western blot shown in a. c Scatterplot showing the negative correlation between the amount of p62 or PU.1, determined by the western blot in a and the autophagic flux (determined in Fig. 2b). d Proximity ligation assay (PLA) showing the interaction between PU.1, LC3-II and p62 in TH9 cells, as well as control antibodies, after 24 h of differentiation. e PLA quantification: number of dots per cell out of 100 cells in two independent experiments. f Immunoblot corresponding to co-immunoprecipitation experiment showing interaction of endogenous PU.1 and p62 in chloroquine-treated TH9 cells (25 μM) after 24 h of differentiation. Shown is a typical experiment out of three. g Immunoblot corresponding to immunoprecipitation performed with anti-PU.1 antibody, followed by western blot detection using poly- and monoubiquitination antibody to examine the presence of poly-ubiquitinated PU.1 in chloroquine-treated TH9 cells (25 μM) after 24 h of differentiation. h Schematic representation of the p62 cDNA fragments used for pull-down experiments. i Synthetic full-length p62 (p62_FL) and p62 lacking the UBA domain (p62_UBA∆) proteins were generated. These two proteins were subsequently incubated with total extracts from TH9 cells differentiated for 24 h. The binding of endogenous PU.1 and LC3-II was tested in a pull-down assay and revealed using western blot analysis. Experiment performed twice. j PLA showing that in chloroquine-treated TH9 cells, PU.1 K63 ubiquitination can be detected after 24 h of differentiation. TH9 cells transfected with control siRNA or p62 siRNA. k p62 inhibition was assessed by western blot 48 h after transfection. l IL-9 expression was analysed by flow cytometry, ELISA and qRT-PCR after 72 h of differentiation. Shown is a typical experiment out of three. *P<0.05; **P<0.01 unpaired Student’s t-test. m, n p62 inhibition and PU.1 protein expression were assessed by western blot 48 h after transfection and quantified. Mean (+s.d.). Shown is a typical experiment out of three. *P<0.05; ***P<0.001 two-way analysis of variance (ANOVA) test