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. Author manuscript; available in PMC: 2017 Nov 14.
Published in final edited form as: Biochemistry. 2017 Oct 18;56(43):5812–5822. doi: 10.1021/acs.biochem.7b00519

Figure 4.

Figure 4

Determination of the RhoPDE transmembrane topology and orientation. Immobilized COS-7 cells were transfected with the dual (C8 and 1D4) epitope-tagged RhoPDE on glass coverslips and incubated with the antibodies under permeabilized or unpermeabilized conditions for immunofluorescent staining, as indicated in the figure and described in Experimental Procedures. Panels A and B show intact and detergent-permeabilized cells, respectively, probed with the 1D4 antibody. Panels C and D show intact and detergent-permeabilized cells, respectively, probed with the C8 antibody. Fluorescence from either probe is observed only in permeabilized cells, indicating both epitopes are intracellular. The COS-7 cells in panels B and D show a diffuse staining pattern typical of proteins localized to the plasma membrane.