Figure 5. Galectin-8 promotes phosphorylation of myosin light chain 2 in a Rho- and ROCK- dependent manner.

A: Gal8 induces phosphorylation of MLC2 in a time-dependent manner. Normal human TM cells were incubated on 100-mm dishes coated with Gal8 for 0.5, 1, and 2 hr. Following incubation, cells were lysed, and protein extracts were subjected to electrophoresis in 12% SDS-PAGE gels. Blots were probed with anti-phosphorylated myosin light chain 2 (ppMLC2) (Thr18/Ser19) antibody. The blots were subsequently stripped and reprobed with anti-MLC antibody. A representative Western blot is shown in the top panel. Images were acquired by Odyssey Infrared Imaging System, and band intensity was quantified by ImageJ (bottom panel). N = 3. B: Phosphorylation of MLC2 is inhibited by Rho and ROCK inhibitors. Normal human TM cells were serum-starved overnight and treated with C3 transferase (2 µg/ml) and Y27632 (20 µM) for 4 hr. Treated cells were detached and plated on Gal8-coated dishes for 2 hr in the presence or absence of inhibitors and were then examined for the expression levels of ppMLC2 as described in the legend to panel A. Top: A representative Western blot; bottom: quantification of ppMLC2. Data are expressed as mean±SEM and analyzed with one-way ANOVA. ***P<0.001 vs control. N = 3.