Fig. 2. The STAT3 Y657* mutant is unstable and goes rapid proteasome-independent decay.

(A) Western blot of extracts from STAT3^−/− A4 cells nontransfected (NT), transfected with expression vectors for STAT3 WT (Myc-tagged), the STAT3 mutant V637M, or the mutant Y657* (both Flag tagged). (B) Mean ± SD of STAT3 (N-ter)/loading control ratio after normalization, for independently performed experiments. Total protein extracts from transfected STAT3^−/− A4 cells with either the WT STAT3 (Myc-tagged) plasmid or the STAT3 mutant V637M (Flag-tagged) (C) or the STAT3 mutant Y657* (Flag-tagged) (D) after treatment with CHX (100 μg/ml). Mean ± SD of STAT3 (N-ter)/loading control ratio after normalization, for independently performed experiments. STAT3 protein T_1/2 (horizontal line) was determined as time after CHX addition until STAT3 levels decreased to 50% of starting level, as indicated by vertical lines. Data are representative of three independent experiments. (E) Total protein extracts from NT or transfected STAT3^−/− A4 cells, after treatment (+) with 20 μM MG-132 for 3 hours. (F) Mean ± SD of STAT3/loading control ratio after normalization, for independently performed experiments. Total extracts of STAT3^−/− A4 cells transfected with either the WT STAT3 (Myc-tagged) plasmid or the STAT3 mutant V637M (Flag-tagged) (G) or the STAT3 mutant Y657* (Flag-tagged) (H), cotransfected with decreasing amounts (50, 25, 10, and 5 ng) of mutant STAT3 plasmid and with constant amounts of WT STAT3 plasmid (50 ng). The transfected cells were treated (+) with 20 μM MG-132 for 3 hours. In all experiments, extracts were probed with antibodies specific for the Myc tag, the Flag tag, and the N-terminal part of STAT3. GAPDH was used as loading control. Mean ± SD of STAT3 (N-ter)/loading control ratio after normalization, for independently performed experiments. Data are representative of three independent experiments. P values by two-way ANOVA followed by Tukey’s multiple comparisons test (G and H).