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. 2013 Aug 1;126(15):3333–3343. doi: 10.1242/jcs.123042

Fig. 3.

Fig. 3.

STAT5a nuclear import is mediated by importin-α3/β1 system. (A) STAT5a-V5 expressed in COS-1 cells was immunoprecipitated using protein G agarose beads, and incubated with bacterially expressed GST-importins in vitro. Importins bound to STAT5a were detected by western blot using anti-GST antibody. The input levels of STAT5a are shown with anti-STAT5a antibody. The lower blot shows 10% of purified GST-importin proteins used in the binding assay. (B) Left: The effect of pooled siRNAs on endogenous importin-α3 or importin-β1 mRNA levels detected by RT-PCR. Pooled siRNAs to importin-α3 or importin-β1 reduced the endogenous respective mRNA levels by ∼80%. Vimentin siRNA was used as a control siRNA. The level of GAPDH was quantified as an internal control. Right: The effect of importin-α3 or importin-β1 siRNAs on the cellular localization of STAT5a-GFP. Nuclear accumulation of STAT5a was inhibited in 10–30% of cultures transiently transfected with importin-α3 or importin-β1 pooled siRNAs, whereas there was no effect with vimentin siRNA.