Figure S5. Further quantifications and... | Rockefeller University Press

Figure S5.

$Further quantifications and in vitro controls. (A–D) Densitometric quantification of GFP-LC3B, LC3B-II, Calnexin, and TOM20 from the density gradient membrane fractions in Figs. 3 and S4 displayed as average ± SD. The intensity of the bands were normalized to the lysate, and statistical significance was assessed by two-way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. WT Basal, n = 3 biological replicates; WT Starved+Baf, n = 4 biological replicates; ATG2 DKO, n = 3 biological replicates. TOM20 signal was oversaturated in fraction 5 of the WT Basal in one replicate, so n = 2 included biological replicates for TOM20 in WT Basal fraction 5. Comparisons are nonsignificant unless noted otherwise in this figure. (E–I) Densitometric quantification of the GFP-LC3B IPs in Fig. 6 comparing protein levels from ATG9A/LC3B-enriched fractions between WT and ATG2 DKO HEK293 cells displayed as average ± SD. The intensity of the bands were normalized to the input, and statistical significance was assessed by two-way ANOVA. All comparisons between WT and ATG2 DKO IPs were nonsignificant. Both cell lines, n = 3 biological replicates, except WT Triton X-100 IPs where n = 2 biological replicates. (J and K) In vitro SMA isolation of GFP-LC3B and LC3B decorated liposomes reveal low frequency of co-residency in SMALPs of randomly distributed proteins. 25 nm liposomes were lipidated as described before (Kauffman et al., 2018), with both LC3B and GFP-LC3B. (J) Coomassie-stained SDS-PAGE gel showing IPs of lipidated liposomes treated with various SMA concentrations or Triton X-100. The 2.5% SMA condition mimics that used on the natural membrane SMA IPs in Fig. 6. The in vitro results show that recovery of two distinct peripheral proteins together is relatively rare and further these results closely mirror the recovery of these two proteins from natural sources as shown with the SMA titrations in Fig. 5 C on levels of GFP-LC3B and LC3B recovery. (K) Densitometric quantification of the LC3B:GFP-LC3B ratio between IPs displayed as average ± SD. Statistical significance was assessed by one-way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. n = 3 in vitro biological replicates.$

Further quantifications and in vitro controls. (A–D) Densitometric quantification of GFP-LC3B, LC3B-II, Calnexin, and TOM20 from the density gradient membrane fractions in Figs. 3 and S4 displayed as average ± SD. The intensity of the bands were normalized to the lysate, and statistical significance was assessed by two-way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. WT Basal, n = 3 biological replicates; WT Starved+Baf, n = 4 biological replicates; ATG2 DKO, n = 3 biological replicates. TOM20 signal was oversaturated in fraction 5 of the WT Basal in one replicate, so n = 2 included biological replicates for TOM20 in WT Basal fraction 5. Comparisons are nonsignificant unless noted otherwise in this figure. (E–I) Densitometric quantification of the GFP-LC3B IPs in Fig. 6 comparing protein levels from ATG9A/LC3B-enriched fractions between WT and ATG2 DKO HEK293 cells displayed as average ± SD. The intensity of the bands were normalized to the input, and statistical significance was assessed by two-way ANOVA. All comparisons between WT and ATG2 DKO IPs were nonsignificant. Both cell lines, n = 3 biological replicates, except WT Triton X-100 IPs where n = 2 biological replicates. (J and K) In vitro SMA isolation of GFP-LC3B and LC3B decorated liposomes reveal low frequency of co-residency in SMALPs of randomly distributed proteins. 25 nm liposomes were lipidated as described before (Kauffman et al., 2018), with both LC3B and GFP-LC3B. (J) Coomassie-stained SDS-PAGE gel showing IPs of lipidated liposomes treated with various SMA concentrations or Triton X-100. The 2.5% SMA condition mimics that used on the natural membrane SMA IPs in Fig. 6. The in vitro results show that recovery of two distinct peripheral proteins together is relatively rare and further these results closely mirror the recovery of these two proteins from natural sources as shown with the SMA titrations in Fig. 5 C on levels of GFP-LC3B and LC3B recovery. (K) Densitometric quantification of the LC3B:GFP-LC3B ratio between IPs displayed as average ± SD. Statistical significance was assessed by one-way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. n = 3 in vitro biological replicates.