Fig. 4.

ARHGEF11 depletion affects MLC activity. (A) Effect of ARHGEF11 depletion on the activity of actomyosin regulators. EpH4 cells transfected with control or ARHGEF11-specific siRNA were analyzed by Western blot by using antibodies to detect phospho-specific or total ERM, MLC, and Src ∼6 h after the calcium switch. Representative data from three independent experiments are shown. (B) Coculture of control and ARHGEF11-depleted EpH4 cells were fixed ∼6 h after the calcium switch and stained for p-MLC and ARHGEF11. Asterisks indicate ARHGEF11-depleted cells. (C) EpH4 cells were treated or not treated with ML-7 in a low calcium medium before the reintroducing normal calcium. The p-MLC was analyzed by Western blot and immunofluorescence after ∼6 h culture in normal calcium medium. (D) The p-MLC level was examined by Western blotting over time in calcium switch. Calcium switch assay was conducted for control cells (a), control cells treated with 30 μM ML-7 in low calcium medium before calcium switch (pretreat) (b), control cells treated with 30 μM ML-7 before and after calcium switch (c), ARHGEF11 knockdown cells (d), and ARHGEF11 knockdown cells treated with 30 μM ML-7 in low-calcium medium before calcium switch (pretreat) (e). Cells were lysed at the indicated time and processed for Western blotting for detection of p-MLC level. Representative data from three independent experiments are shown. (E) TER was measured at the indicated times (data presented as means ± SD; n = 3). The y axis is the percentage of the maximal TER measured in this experiment. (F) Immunostaining for p-MLC in cells 60 h after calcium switch. (Scale bars: 10 μm.)