Fig. 4
USP24-stabilized p300 increases NF-κB and IL-6 expression in M2 macrophages. a Western blotting was used to analyze the indicated protein levels in scramble- and USP24-knockdown M2 macrophages by using two different shRNA clones. b Q-PCR was used to analyze IL-6 RNA level in scramble- and p300-knockdown M2 macrophages. c IL-6 level in conditioned medium derived from scramble- and p300-knockdown M2 macrophage was studied by ELISA (n = 4). d The recruitment of acetyl histone H3 to the promoters of IL-6 and NF-κB in USP24-knockdown M2 macrophages was analyzed by chromatin immunoprecipitation (IP) assay. e The lysates were harvested from M2 macrophages for immunoprecipitation with anti-USP24 antibodies. IP samples were used to study the USP24 and p300 levels by western blotting with anti-USP24 and anti-p300 antibodies. f The lysates were harvested from scramble- and USP24-knockdown M2 macrophage for immunoprecipitation with anti-p300 antibodies. IP samples were used to study the ubiquitinated p300 by western blotting with anti-ubiquitin antibody. g The lysates collected from scramble- or USP24-knockdown M2 macrophages with MG132 treatment were used to study the indicated protein levels by western blotting. h Scramble and USP24-knockdown M2 macrophages were treated with CHX and collected at indicated time points. p300 protein stability was analyzed by western blotting. i, j NF-κB protein and mRNA levels were measured in scramble- and USP24-knockdown M2 macrophages by western blotting, RT-PCR (i) and Q-PCR (j). k, l Western blotting (k) and Q-PCR (l) were used to analyze indicated protein and mRNA levels in scramble- and p300-knockdown M2 macrophages. m The lysates were harvested from A549 cells with or without USP24 knockdown or p300 overexpression and analyzed by using the western blotting and RT-PCR. Protein and mRNA levels were quantified after independent experiments (n = 3). Results of western blot and Q-PCR were normalized with actin, tubulin or GAPDH and expressed as fold of control. Data are shown as mean ± SEM, two-tailed unpaired Student’s t-test, *P < 0.05, **P < 0 .01, and ***P < 0.005
