Fig. 3. Intracellular distribution of amphiphysin 2 and SNX4. (A-O) HeLa cells were transiently transfected (A,D) with Amp2m in pRK5myc, (B,E) with Bramp2 in pRK5myc or (C,F) with SNX4 in pRK5myc. They were (A-C) directly fixed with PFA, stained with anti-myc antibody and fluorochrome-conjugated secondary antibodies or (D-F) first pre-permeabilized with 0.01% saponin before fixation in PFA and staining as above. (G-L) HeLa cells were co-transfected with Amp2m in pRK5 plus SNX4 in pRK5myc, pre-permeabilized with 0.01% saponin before co-staining with anti-BIN1 mouse monoclonal antibody and anti-myc rabbit polyclonal antibody, followed by species-specific secondary antibody. (M-O) Hela cells were co-transfected with Bramp2m in pRK5 plus SNX4 in pRK5myc, pre-permeabilized with 0.01% saponin before co-staining. (J,K,L) panels are magnifications of the (G,H,I) panels, respectively. (P) native 3T3 cells. (Q-R) 3T3 cells were transiently transfected with SNX4 in pRK5myc. (P-R) cells were pre-permeabilized in 0,01% saponin before fixation and staining with anti-myc plus rabbit anti-amphiphysin 2 antibody, followed by fluorochrome conjugated secondary antibodies.

Images were acquired on a confocal microscope and pseudo-colored with Metamorph. Co-localization appears in yellow.

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