Figure S4. Additional considerations for... | Rockefeller University Press

Figure S4.

$Additional considerations for ATG9A and LC3B distribution and co-residency. (A) Immunoblot of cell membrane fractions from GFP-LC3B expressing WT HEK293 cells showing enrichment of ATG9A and LC3B together in fraction 5 and 6. Cells were grown in DMEM under basal conditions like the KO cells lines in Fig. 4. Loaded protein: 3 µg. (B) Densitometric quantification of ATG9A, GFP-LC3B, LC3B-II, Calnexin, and TOM20 in WT Basal membrane fractionation displayed as average ± SD. The intensity of the bands in A were normalized to the lysate, and statistical significance was assessed by two-way ANOVA. **, adjusted P value <0.01. ****, adjusted P value <0.0001. n = 3 biological replicates, except TOM20 signal was oversaturated in fraction 5 in one replicate, so n = 2 included biological replicates for TOM20 in fraction 5. (C) Immunoblot comparing endogenous levels of LC3B-II in HEK293 cells with or without overexpression of GFP-LC3B. The endogenous LC3B pool in ATG2 DKO cells is mostly but not completely in form II, perhaps because the ability to lipidate LC3B is saturated with GFP-LC3B overexpression. WT and ATG2 DKO samples are from the same blot. Loaded protein: 10 µg. (D) Immunoblots from membrane fractions from both WT and ATG2 DKO HEK293 cells were run on the Bolt SDS-PAGE system, which allows larger fusion proteins to run far enough to separate protein conjugated to lipid, to distinguish GFP-LC3B-I and GFP-LC3B-II. Colored dots indicate whether the non-lipidated (blue) and/or lipidated (red) forms of GFP-LC3B were detectable in each lane. Loaded protein: 3 µg. (E) Immunoblot of sequential IPs as in Fig. 6 F, first with GFP-LC3B and then with endogenous ATG9A, from GFP-LC3B expressing WT HEK293 cells under basal conditions. After the first IP, the sample was treated with 6 M GdnHCl before diluting over 25X and performing a second IP against ATG9A. Recovery of GFP-LC3B after the second IP is more robust in SMALPs than in soluble Triton-X micelles. Input protein for GFP IPs: 50 µg. Input for ATG9A IPs: 98% of the material collected from the GFP IPs. Loaded input protein: 5 µg (10%). Loaded GFP IPs: 1% of the total collected from the beads. Loaded ATG9A IPs: 50% of the total collected from the beads after GdnHCl treatment. (F) Immunoblot of sequential IPs as in Fig. 6 F, using WT HEK293 cells expressing both GFP-LC3B and FLAG-ATG9A. After the first IP, the sample was treated with 6 M GdnHCl before diluting over 25X and performing a second IP against FLAG. Recovery of GFP-LC3B after the second IP is more robust in SMALPs than in soluble Triton-X micelles. Input protein for GFP IPs: 35 µg. Input for FLAG IPs: 98% of the material collected from the GFP IPs. Loaded input protein: 0.35 µg (1%). Loaded GFP IPs: 1% of the total collected from the beads. Loaded ATG9A IPs: 50% of the total collected from the beads after GdnHCl treatment.$

Additional considerations for ATG9A and LC3B distribution and co-residency. (A) Immunoblot of cell membrane fractions from GFP-LC3B expressing WT HEK293 cells showing enrichment of ATG9A and LC3B together in fraction 5 and 6. Cells were grown in DMEM under basal conditions like the KO cells lines in Fig. 4. Loaded protein: 3 µg. (B) Densitometric quantification of ATG9A, GFP-LC3B, LC3B-II, Calnexin, and TOM20 in WT Basal membrane fractionation displayed as average ± SD. The intensity of the bands in A were normalized to the lysate, and statistical significance was assessed by two-way ANOVA. **, adjusted P value <0.01. ****, adjusted P value <0.0001. n = 3 biological replicates, except TOM20 signal was oversaturated in fraction 5 in one replicate, so n = 2 included biological replicates for TOM20 in fraction 5. (C) Immunoblot comparing endogenous levels of LC3B-II in HEK293 cells with or without overexpression of GFP-LC3B. The endogenous LC3B pool in ATG2 DKO cells is mostly but not completely in form II, perhaps because the ability to lipidate LC3B is saturated with GFP-LC3B overexpression. WT and ATG2 DKO samples are from the same blot. Loaded protein: 10 µg. (D) Immunoblots from membrane fractions from both WT and ATG2 DKO HEK293 cells were run on the Bolt SDS-PAGE system, which allows larger fusion proteins to run far enough to separate protein conjugated to lipid, to distinguish GFP-LC3B-I and GFP-LC3B-II. Colored dots indicate whether the non-lipidated (blue) and/or lipidated (red) forms of GFP-LC3B were detectable in each lane. Loaded protein: 3 µg. (E) Immunoblot of sequential IPs as in Fig. 6 F, first with GFP-LC3B and then with endogenous ATG9A, from GFP-LC3B expressing WT HEK293 cells under basal conditions. After the first IP, the sample was treated with 6 M GdnHCl before diluting over 25X and performing a second IP against ATG9A. Recovery of GFP-LC3B after the second IP is more robust in SMALPs than in soluble Triton-X micelles. Input protein for GFP IPs: 50 µg. Input for ATG9A IPs: 98% of the material collected from the GFP IPs. Loaded input protein: 5 µg (10%). Loaded GFP IPs: 1% of the total collected from the beads. Loaded ATG9A IPs: 50% of the total collected from the beads after GdnHCl treatment. (F) Immunoblot of sequential IPs as in Fig. 6 F, using WT HEK293 cells expressing both GFP-LC3B and FLAG-ATG9A. After the first IP, the sample was treated with 6 M GdnHCl before diluting over 25X and performing a second IP against FLAG. Recovery of GFP-LC3B after the second IP is more robust in SMALPs than in soluble Triton-X micelles. Input protein for GFP IPs: 35 µg. Input for FLAG IPs: 98% of the material collected from the GFP IPs. Loaded input protein: 0.35 µg (1%). Loaded GFP IPs: 1% of the total collected from the beads. Loaded ATG9A IPs: 50% of the total collected from the beads after GdnHCl treatment.