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. 2016 Dec 20;292(5):1637–1647. doi: 10.1074/jbc.M116.755546

FIGURE 2.

FIGURE 2.

The effect of ACTN4 on GR-mediated transcriptional activation. A, HEK293T cells were treated with (+) or without (−) dexamethasone (Dex, 100 nm) for 8 h. Whole cell extracts were prepared and immunoprecipitated (IP) with anti-GR antibodies. Proteins were detected by Western blotting (WB) using anti-ACTN4 and anti-GRα antibodies as indicated. B, HeLa cells were transfected with an HA-GRα expression plasmid, and lysates were prepared and incubated with immobilized, bacterially expressed GST-ACTN4 fusion protein in the presence of dexamethasone. Pulldown fractions were subjected to Western blotting with HA antibodies. C, HEK293T cells were cotransfected with a GRE-luciferase reporter plasmid and HA-ACTN4 expression plasmid. The cells were then treated with dexamethasone for 12 h, and luciferase activity was measured. D, HPC whole cell extracts were prepared and immunoprecipitated with anti-ACTN4 antibodies and anti-HA antibodies as a control. Proteins were detected by Western blotting using anti-ACTN4 and anti-GR (arrowhead) antibodies as indicated. E, mouse glomerular extracts were prepared and immunoprecipitated with anti-GR or anti-HA antibodies, followed by Western blotting with anti-ACTN4 or GR antibodies. F, ACTN4 was stably knocked down by shRNAs in HPCs. shCtrl was used for comparison. A GRE-luciferase reporter plasmid and an HA-GR expression plasmid were transfected into shCtrl and shACTN4 HPCs, treated with vehicle (Veh) or dexamethasone for 12 h, and the reporter activity was analyzed. G, HDAC7 was stably knocked down by shRNAs in HPCs. shCtrl was used for comparison. A GRE-luciferase reporter plasmid and an HA-GR expression plasmid were transfected into shCtrl and shHDAC7 HPCs and treated with Veh or dexamethasone for 12 h, and the reporter activity was analyzed. H, HPCs were transiently transfected with ACTN4 (32), ACTN4 (FL, Δ831–869), ACTN4 (Iso), or ACTN4 (Iso, Δ441–479) and a GRE-Luciferase reporter plasmid and treated with or without dexamethasone, and reporter activity was measured. ***, p < 0.001; n.s., not significant.