To study the molecular mechanism by which nonmuscle myosin II (MII) regulates protrusion and adhesion dynamics in migrating cells, NIH3T3 cells were co-stained for βPIX (green) and myosin IIA (red). As a member of the Dbl family of Rho-GEFs (guanine nucleotide exchange factors), βPIX could contribute to cell motility through interactions with myosin. Findings from this study help elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells. This image is original data from Fig. S2, "Colocalization of MII and GEFs in NIH3T3 fibroblasts," in J. Cell Biol. 2010. Vol. 190(4):663–674
Cells were cultured in DME (Invitrogen) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen) at 37°C in a humidified 5% CO2 incubator. Cells were fixed using 3.7% paraformaldehyde in PBS for 15 min, permeabilized using 0.2% Triton X-100 in PBS for 2 min, blocked with 2% BSA in PBS, and stained with the indicated primary antibodies for at 4°C overnight, followed by incubation with a secondary Alexa Fluor 488–, 546– or 594–conjugated antibody. Images were captured by Leica TCS SP2 confocal microscope with HCX PL APO 63X objective.
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