Figure 5. Styrene maleic acid (SMA)... | Rockefeller University Press

Figure 5.

$Styrene maleic acid (SMA) copolymer nanodiscs capture ATG9A and LC3B together in nanoscale membranes. (A) Transmission electron microscopy (TEM) of 200 nm liposomes treated with varying concentrations of SMA copolymer. The titration reveals the formation of SMA lipid particles (SMALPs) even at low SMA concentrations, with complete solubilization of liposomes at 1.5–2.5% SMA. Scale bar in first panel: 200 nm. Scale bars in SMA treated panels: 100 nm. (B) Cartoon of the transmembrane domains of the ATG9 trimer in a SMALP beside a lipidated LC3B. (C) Immunoblot of the bulk membrane fraction from GFP-LC3B expressing ATG2 DKO HEK293 cells. SMA was titrated in leading to increasing solubilization of the material, and then GFP-LC3B was isolated by immunoprecipitation (IP). ATG9A and GFP-LC3B co-IP with little to no membrane contaminants at higher concentrations of SMA. At lower concentrations of SMA, where membranes are incompletely solubilized into nanodiscs, the IP continues to pull-down all components. Input protein for each IP: 200 µg. Loaded input protein: 3 µg (1.5%). Loaded IPs: 5% of the total collected beads. (D) Immunoblot of the separation of intact membranes from SMALPs via density gradient fractionation (cartoon above) after treatment with a low concentration of SMA (0.1%) on the bulk membrane fraction from GFP-LC3B expressing ATG2 DKO HEK293 cells. Input protein on gradient: 600 µg. Input for each IP: 90% of the material collected from the gradient. Loaded pre-SMA input protein: 3 µg (0.5%). Loaded gradient protein: 5% of the total collected. Loaded IPs: 10% of the total collected beads.$

Styrene maleic acid (SMA) copolymer nanodiscs capture ATG9A and LC3B together in nanoscale membranes. (A) Transmission electron microscopy (TEM) of 200 nm liposomes treated with varying concentrations of SMA copolymer. The titration reveals the formation of SMA lipid particles (SMALPs) even at low SMA concentrations, with complete solubilization of liposomes at 1.5–2.5% SMA. Scale bar in first panel: 200 nm. Scale bars in SMA treated panels: 100 nm. (B) Cartoon of the transmembrane domains of the ATG9 trimer in a SMALP beside a lipidated LC3B. (C) Immunoblot of the bulk membrane fraction from GFP-LC3B expressing ATG2 DKO HEK293 cells. SMA was titrated in leading to increasing solubilization of the material, and then GFP-LC3B was isolated by immunoprecipitation (IP). ATG9A and GFP-LC3B co-IP with little to no membrane contaminants at higher concentrations of SMA. At lower concentrations of SMA, where membranes are incompletely solubilized into nanodiscs, the IP continues to pull-down all components. Input protein for each IP: 200 µg. Loaded input protein: 3 µg (1.5%). Loaded IPs: 5% of the total collected beads. (D) Immunoblot of the separation of intact membranes from SMALPs via density gradient fractionation (cartoon above) after treatment with a low concentration of SMA (0.1%) on the bulk membrane fraction from GFP-LC3B expressing ATG2 DKO HEK293 cells. Input protein on gradient: 600 µg. Input for each IP: 90% of the material collected from the gradient. Loaded pre-SMA input protein: 3 µg (0.5%). Loaded gradient protein: 5% of the total collected. Loaded IPs: 10% of the total collected beads.