Integrin-Ligand Interactions Control Migration Figure 3. Effect of myosin II on protrusion. (A) CHO.B2 cells transfected with the indicated fluorescently tagged integrin subunit(s) were platted on the indicated substrate for 1 hour, then blotted for MHC IIA, MHC IIB, phosphorylated (p) and total RLC. pRLC does not increase noticeably on ICAM-1 but does on both FN and LN in a dose dependent manner. Note also that MHC IIA and MHC IIB do not change with substrate concentration. Also, the densitometric quantification of pRLC corrected for load using total RLC is shown under each blot. At least three experiments were quantified for each substrate. (B) Adhesion on fibronectin (a5b1) or laminin (a6b1) determines the subcellular distribution of pRLC (Ser19). CHO.B2 cells were (top) transfected with a5-GFP and plated for 60 min on FN (2 mg/ml); (bottom) CHO B2 cells transfected with a6-GFP and plated for 60 min on LN (10 mg/ml). The cells were stained for paxillin and phosphorylated (Ser19) RLC as indicated. Note the more fibrillar distribution of the pRLC in the cells on FN. Scale bar = 10 mm. (C) Over-expression of MHC IIA, but not MHC IIB, inhibits protrusion of CHO.B2 cells on FN but not on LN. CHO.B2 cells were doubly transfected with a5- or a6-GFP and mCherry-MHC IIA or MHC IIB as indicated, then plated on the corresponding substrate (FN for a5, LN for a6). Scale Bar = 10 mm. Protrusion rates from 4 minute movies were calculated from kymographs and plotted. Data are expressed as the mean 6 SD of at least 3 independent experiments. (Protrusion number = 7, 7, 12, 12, 10, 11, respectively.) P,0.001 for cells on FN expressing ectopic MHC IIA compared to cells expressing ectopic MHC IIB or control cells. doi:10.1371/journal.pone.0040202.g003 PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40202