Fig. 7
The role of β-TrCP in USP24 knockdown increased DNMT1 and IκB. a Indicated protein levels were examined in scramble knockdown and USP24-knockdown A549 cells by western blotting (n = 3). b Scramble- and USP24-knockdown A549 cells were treated with CHX and IκB protein stability was analyzed by western blotting (n = 3). c Scramble- and USP24-knockdown A549 cells were overexpressed with myc-ubi and cells were collected for immunoprecipitation with anti-myc antibody. IP samples were analyzed by western blotting. d Scramble-knockdown and USP24-knockdown A549 cells were overexpressed with β-TrCP and collected for analyzing indicated proteins by western blotting (n = 3). Results of western blot were normalized with actin and expressed as fold of control. e Indicated proteins and mRNAs levels in A549 cells with or without USP24 knockdown or β-TrCP overexpression were analyzed by western blotting and RT-PCR. f GFP and GFP-USP24 overexpressed A549 cells were fixed for immunofluorescence assay with indicated antibodies. GFP or GFP-USP24 overexpressed cells were indicated with white arrowheads. Yellow arrowheads indicate GFP-USP24 negatively expressed cells in GFP-USP24 overexpression group. Scale bar represents 10 μm. g Luciferase activity was measured by transfecting NF-κB response element (RE) containing vector into scramble knockdown, USP24 knockdown, GFP overexpressed, and GFP-USP24 overexpressed A549 cells (n = 3). Results were normalized with control and expressed as fold of control. h, i The intersections between 452 NF-κB target genes (blue circle), 125 USP24 upregulated genes (red circle) and 251 USP24 downregulated genes (green circle) were analyzed (h). Heatmaps of intersected were shown, and metastatic genes were indicated by asterisks (i). Data are shown as mean ± SEM, two-tailed unpaired Student’s t-test, ns for not significant, *P < 0.05 and ***P < 0.005
