Figure 5
Membrane docking of Smad3 is endocytosis-independent.
(a,b) Diffusion rates (D) of membrane-docked EGFP-Smad3 molecules with the marked D value before (a) and after (b) TGF-β1 stimulation in HeLa cells expressing K44A-dynamin2-HA to inhibit endocytosis. The two groups of D values are significantly different (p < 0.001, Mann-Whiteney U test). (c,d) Cumulative histograms (solid) of the on-times of EGFP-Smad3 molecules before (c) and after (d) TGF-β1 stimulation in HeLa cells expressing K44A-dynamin2-HA to inhibit endocytosis. The histograms were fitted with a single exponential function. The dotted lines are the fitting curves with the time constants τu (0.70 s) and τs (0.82 s). The correlation coefficients of the fitting lines were both above 0.97. The two groups of τ values are significantly different (p < 0.001, Mann-Whiteney U test). (e) The effect of endocytosis on Smad3 phosphorylation and nuclear transportation shown by confocal microscopy. HeLa cells expressing K44A-dynamin2-HA were immunostained with antibodies against HA and p-Smad3. Confocal imaging of p-Smad3 (pAb, Alexa-488) and K44A-dynamin2-HA (HA mAb, Alexa-647) before and after TGF-β1 stimulation indicated that the nuclear accumulation of p-Smad3 was not dependent on endocytosis. Scale bars: 5 μm. (f) Western blotting of K44A-dynamin2-HA and p-Smad3. GAPDH was used as the internal control.
