GC interaction increases the levels of phosphorylated MLC at the apical junction and apical membrane.

<p>(A-E) Polarized T84 cells were untreated or pre-treated with the ROCK inhibitor Y27632 (Y) and the MLCK inhibitor ML-7 (M) and PIK, and apically incubated with piliated and non-piliated MS11Opa+ and ΔOpa for 6 h in the presence or absence of inhibitors. Cells were stained for phosphorylated MLC (pMLC) and GC and analyzed using 3D-CFM. FIRs of pMLC at the apical to lateral region (A-C) and at the junctional to non-junctional region (D and E) were determined. Shown are representative xz (A) and xy images at the apical junctional location (D), FI maps (D, right panels), and the average FIR (±SD) (B, C, and E) of >50 cells from three independent experiments. Arrows indicate GC. Bar, 5 μm. (F-H) Polarized T84 cells were treated with inhibitors and incubated with piliated GC as above. Cells were then lysed and analyzed by Western blot probing for MLC, pMLC and β-tubulin. The blot was quantified by Phosphorimager. Shown are representative blots (F) and the average fold of increase (±SD) in pMLC:MLC (G) and MLC:tubulin (H) ratios over no GC control from three independent experiments. ***<i>p</i> ≤0.001; **<i>p</i> ≤ 0.01; *<i>p</i>≤0.05.