File S1 - Studying the Nucleated Mammalian Cell Membrane by Single Molecule Approaches

Supporting text and Figures S1–S11. Figure S1. The distribution of Na+-K+ ATPase in the inner leaflets of human erythrocyte membranes. Na+-K+ ATPase was labeled with Na+-K+ ATPase antibody conjugated with cy5, and the fluorescence image was acquired with STORM. There are a plenty of Na+-K+ ATPases in the inner leaflet membrane, and the majority of the proteins form microdomains. Scale bar: 2 µm. Figure S2. Localizing the EGFR on the outer surface of A549 cells by topography and recognition imaging (TREC). The cell was gently fixed by 4% paraformaldehyde before imaging. EGFR was localized on the surface of A549 cells by scanning the cells with EGF modified AFM tips. (A) The topography of the cell surface shows a relatively smooth feature without protein domain. (B) The corresponding recognition imaging to show the location of EGFRs (dark areas), which indicates that EGFRs exist in the microdomains (about hundreds of nanometers). Scale bar: 500 nm. Figure S3. Digestion of the outer leaflet of MDCK cell membranes by PNGase F. The outer leaflet of membranes was treated with PNGase F, which can cleave most of saccharides from glycoproteins. (A) The topography of the outer leaflet membrane treated by PNGase F. There is no pit or indent visible on the smooth membrane. (B) Cross section analysis along the green line in (A), which shows no apparent decrease of the thickness of membranes. Scale bar: 150 nm. Figure S4. The outer leaflet membrane of a primary hepatocyte prepared from rat liver. The outer surface is pretty smooth as MDCK cells (Fig.1). Scale bar: 300 nm. Figure S5. The outer and inner leaflet membrane of erythrocytes from crucian carp. (A) The outer leaflets of membranes of red blood cell membrane from crucian carp. (B) A whole inner leaflet of red blood cell membrane from crucian carp. There are dense proteins in the inner leaflet membrane. (C) The magnified image from (B). Scale bars: 200 nm in (A), 4 µm in (B), 1 µm in (C). Figure S6. The morphology of the outer and inner leaflet of human platelets. (A) The morphology of the outer leaflet of a platelet. (B) The inner leaflet membrane is rough with a plenty of proteins. The proteins are in the status of dispersed domains, which can be clearly observed in the magnified image (C). Scale bars: 100 nm in (A), 1 µm in (B), 500 nm in (C). Figure S7. The membranes of mitochondrion from rat liver. (A) The intermembrane space surface of the inner mitochondrial membrane. The membrane surface is very smooth with the roughness of 0.6±0.2 nm. (B) The matrix side of the inner mitochondrial membrane. There are a plenty of proteins in the inner mitochondrial membrane, and they tend to form microdomains. Scale bars: 150 nm in (A), 200 nm in (B). Figure S8. The membranes of Golgi apparatus from Hela cells. (A) The smooth outer leaflet membrane of Golgi apparatus. (B) The inner leaflets of membranes are covered with proteins that tend to form dispersed microdomains. Scale bars: 150 nm in (A), 200 nm in (B). Figure S9. Western blot analysis of protein differential distribution in Hela cells. (A) Hela cells treated with PBS (ctrl), protease mixture and 0.1% Triton X-100/protease mixture was used as samples. After electrophoresis CD47 monoclonal antibody B6H12 was used as marker for amino acid at the outer membrane leaflets. Compared with control, CD47 band significantly decreased in protease mixture treated sample. Bands of CD47 and actin both disappeared when 0.1% Triton X-100 and protease mixture double treatments were applied. (B) Membrane fraction (mem) or intact Hela cells (total) were used as samples. Band 3 polyclonal antibody targeted to the intracellular N-terminal serves as markers for amino acid at the inner membrane leaflets. The intensity of Band3 was much stronger in the membrane fraction which implied more epitopes were exposed. Figure S10. Topology model of CD47 (Brown and Frazier, 2001). Figure S11. Topology model of human erythrocyte BandIII (Bonar and Casey, 2008)
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