The SNX-PX-BAR Family in Macropinocytosis: The Regulation of Macropinosome Formation by SNX-PX-BAR Proteins
Figure 4
SNX1, SNX5, SNX9 and SNX18 associate with early macropinosomes.
A: HEK-Flp-In cells were pulsed with 100 µg/mL dextran (10,000 MW) conjugated to Alexa-647 (dextran-647) for 5 minutes before being transferred to 4°C and washed with 0.45 mM CaCl2 1 mM MgCl2 PBS. The cells were then treated with 80 U/mL Streptolysin O for 5 minutes at 4°C to only permeabilize the plasma membrane before washing in 0.45 mM CaCl2 1 mM MgCl2 PBS and incubating with 37°C PBS for 5 minutes. Following fixation with 4% PFA for 30 minutes at 4°C, cell monolayers are incubated with monoclonal and polyclonal antibodies against SNX1 and SNX5 respectively, followed by Alexa-488-conjugated goat-anti-mouse and Cy3-conjugated goat-anti-rabbit IgG secondary antibodies. B, C, and D: 24 hours post transfection, HEK-Flp-In cells transfected with pEGFP-SNX9 (B), pEGFP-SNX18 (C) and pEGFP-SNX33 (D) were pulsed with dextran-TR for 5 minutes at 37°C prior to fixation at 4°C in 4% PFA. Images were collected on an LSM 510 Meta confocal microscope. Scale bar = 5 µm.