Supporting Information S1 - Stimulus-Specific Activation and Actin Dependency of Distinct, Spatially Separated ERK1/2 Fractions in A7r5 Smooth Muscle Cells

Co-localization of phospho-ERK1/2 with filamentous actin and ERK1/2 distibution between caveolar and non-caveolar fractions. (A) A7r5 cells were stimulated with either DPBA or FCS for 10 minutes, or left unstimulated, and then fixed and stained for immunofluorescence microscopy with a phospho-ERK1/2 antibody. Cells were co-stained with phalloidin to visualize actin filaments and with DAPI to visualize nuclei. Please note the filamentous ERK1/2 staining after DPBA stimulation (E and H, arrowheads), and the stronger nuclear ERK1/2 staining after FCS stimulation (I and L, arrows) compared to DPBA stimulation (E and H, arrows). Scale bar, 20 µM. (B) Unstimulated A7r5 lysates were subjected to density gradient ultracentrifugation, and fractions (numbered from top to bottom) were analyzed for total ERK1/2 in western blots. The caveolar fractions were determined by co-staining the membranes with a caveolin-1 antibody. The percentage of ERK1/2 in the caveolar fraction was determined by densitometry (n = 3). (PDF)