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

FIGURE 4.

FIGURE 4.

The effect of the ACTN4 LXXAA mutation on potentiating GR-mediated transcriptional activity. A and B, expression plasmids for ACTN4 (WT), ACTN4 (Iso) (A), or an ACTN4 mutant (LXXAA) (B) were co-transfected with a GR expression plasmid and GRE-luciferase reporter plasmid in HEK293T cells, followed by treatment with Veh or dexamethasone (Dex) for 12 h, and the reporter activity was analyzed. C, HeLa cells were transfected with an HA-GR expression plasmid, and lysates were prepared and incubated with bacterially expressed GST-ACTN4 (WT) or GST-ACTN4 (LXXAA) fusion proteins in the absence or presence of dexamethasone (100 nm). Pulldown fractions were subjected to Western blotting with HA antibodies. D, the LXXAA mutation does not affect ACTN4 F-actin filament binding activity. Recombinant GST, GST-ACTN4 (WT), and GST-ACTN4 (LXXAA) were purified and subjected to F-actin co-sedimentation assays. S, soluble fraction; P, pellet fraction. Note that the differences of GST-ACTN4 in the pellet fractions between lanes 2 and 4 and lanes 6 and 8 are similar.