Fig. 4. Subcellular distribution of CD4, myc-TRPC4 and myc-TRL in HEK293 cells co-expressing YFP-EBP50. (A-F) Non-permeabilized cells fixed with PFA. (G-L) Cells permeabilized with Triton X-100 after fixation. (M-R) Cells pre-extracted with digitonin before fixation. Upper panels: cells co-expressing YFP-EBP50 (A,G,M) and CD4 (B,H,N). Middle panels: cells co-expressing YFP-EBP50 (C,I,O) and myc-TRPC4 (D,J,P). Lower panels: cells co-expressing YFP-EBP50 (E,K,Q) and myc-TRL (F,L,R). The arrows in L and R point to regions of myc-TRL accumulation. To detect the transmembrane proteins, the transfected cells were probed with an anti-CD4 (upper panels) or an anti-myc (middle and lower panels) antibody and then stained with an Alexa 594-conjugated secondary antibody. Images were collected with a 100x oil immersion objective and filter sets for FITC (to detect YFP-EBP50) and Texas Red (to detect CD4 and the myc-tagged channels), using identical settings. Bar, 20 µm.