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. 2014 Oct 21;289(49):33904–33915. doi: 10.1074/jbc.M114.618405

FIGURE 2.

FIGURE 2.

hFBXO22a does not affect SR turnover or targeting to the proteasome. A, SR ubiquitination (Ubiq) is not increased when cotransfected with FBXO22a. HA-SR was cotransfected with Myc-hFBXO22a or GFP and incubated with or without MG132 (a proteasome inhibitor). HA immunoprecipitate (IP) was probed with mouse anti-FLAG (1:500, Sigma) to reveal ubiquitin conjugates (top panel). Non-ubiquitinated SR was revealed with mouse anti-HA (1:1000, bottom panel). B, SR turnover is unaffected by hFBXO22 overexpression. HA-SR was cotransfected with hFBXO22a, hFBXO22b, or GFP (n = 3). Cells were incubated in a medium containing [35S]methionine/cysteine and chased for the indicated times (top panel). Bottom panel, PhosphorImager analysis of [35S]SR. C, endogenous FBXO22a knockdown does not affect SR ubiquitination. HEK293 cells were cotransfected with HA-SR, FLAG-ubiquitin, siRNA to hFBXO22a, or control (Ctl) siRNA, and ubiquitination was revealed as in Fig. 2A after 12 h incubation with 30 μm MG132. Bottom panel (input), successful knockdown of endogenous hFBXO22a expression revealed with mouse anti-FBXO22 (1:1000). D, knockdown of endogenous hFBXO22a does not affect endogenous SR steady-state levels in A172 glioblastoma cells (top panel). Transfection was performed with siRNA against hFBXO22a, which down-regulated hFBXO22a expression (center panel). Bottom panel, β-actin loading control. The graph shows the lack of change in the steady-state levels of endogenous SR from seven independent experiments. a.u., arbitrary units; ns, not significant. E, SR preferentially interacts with free FBXO22a species. Myc-SR or Myc-KDM4A was cotransfected with HA-FBXO22a and immunoprecipitated with anti-Myc. Cul1 was present in the Myc-KDM4A/HA-FBXO22a coimmunoprecipitate but absent from the Myc-SR·HA-FBXO22a complex (top panel). HA-FBXO22a was revealed with mouse anti-HA (1:1000, center panel), whereas immunoprecipitated Myc-KDM4A or Myc-SR was revealed with mouse anti-Myc (1:1000, Sigma, bottom panel). F and G, HA-FBXO22a, but not HA-SR, interacts with ectopically expressed Cul1 (F) or Skp1a (G). H, FBXO22a, KDM4A, and H3K9Me3 levels are unchanged in brain homogenates of SR-KO mice. Cyt, brain cytosolic fraction; PN, brain purified nuclear fraction. The blots are representative of at least three different experiments.