Smad2/3 nuclear translocation is inhibited by K
3.1 channel blockade. (A) The ratio of nuclear to whole cell staining of Smad2/3 demonstrates a significant increase in nuclear translocation following TGFβ1 stimulation compared to control. TRAM-34 (200 nM) inhibited TGFβ1-induced Smad2/3 nuclear translocation (NFC n = 4, IPF n = 4) whereas the structurally related molecule TRAM-85 without KCa3.1 blocking properties did not inhibit nuclear translocation. NFC and IPF data were pooled for statistical analysis. (B) Representative fluorescent microscopy images illustrate the increased expression of Smad2/3 following TGFβ1 stimulation and its movement into the nucleus, which was significantly attenuated by TRAM-34 (200 nM). (C) ICA-17043 (100 nM) also significantly attenuated Smad2/3 nuclear translocation, which can be seen visually in (D). (E) Quantification of Western blot analysis confirms that the total Smad2/3 in the nuclear enriched fraction is significantly increased following TGFβ1 stimulation and attenuated by ICA-17043 (100 nM). Results are normalized to TATA Binding Protein (TBP) and representative Western blot analysis images are shown. (F) Similarly, the total Smad2/3 was examined in the cytoplasmic-enriched fraction; however, no changes were found following TGβ1 stimulation or treatment with ICA-17043 (NFC n = 2 and IPF n = 3, data pooled). Results are represented as mean ± SEM ***P < 0.001, **P < 0.01 (two-way ANOVA corrected by Sidaks multiple comparison test), ##
P < 0.0001, one sample t test, #
P < 0.05, paired t test.