Skip to main content
. 1999 Nov 29;147(5):1023–1038. doi: 10.1083/jcb.147.5.1023

Figure 3.

Figure 3

Figure 3

Figure 3

Rho- and Rho-kinase–dependent phosphorylation of MBS at Ser-854 in MDCK cells. (A) TPA- and HGF-induced phosphorylation of MBS at Ser-854. Serum-deprived MDCK cells were stimulated with 200 nM TPA, 50 pM HGF for 15 min or 5 mM dibutyryl cAMP (cAMP) for 30 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). Arrowhead indicates the position of MBS phosphorylated at Ser-854. A minor band with apparent relative molecular mass of 90 kD may be a degradation product of MBS. (B) Time course of the phosphorylation of MBS at Ser-854 in the TPA- and HGF-stimulated MDCK cells. Serum-deprived MDCK cells were stimulated with 200 nM TPA (a) or 50 pM HGF (b) for 3, 5, 15, 30, 60, or 120 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The amount of MBS phosphorylated at Ser-854 was quantitatively determined by scanning densitometry. The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab at 0 min was set at 100 arbitrary units. The values shown are means ± SE of triplicates. (C) Inhibition of the TPA- and HGF-induced MBS phosphorylation by C3 or Rho-kinase inhibitors. Nonpretreated (2), 50 or 100 μg/ml C3-pretreated (3 and 4), 1 or 10 μM of HA1077-pretreated (5 and 6), or 1 or 10 μM of Y-32885–pretreated (7 and 8) serum-deprived MDCK cells were stimulated with (2–8) or without (1) 200 nM TPA (a) or 50 pM HGF (b) for 15 min and the lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab in the nonstimulated cells was set at 100 arbitrary units. The values shown are means ± SE of triplicates.