![ATG9A and LC3B reside in the same membrane fractions enriched for autophagosomes or their precursor membranes. (A) Experimental scheme for density gradient membrane fractionation protocol (see Materials and methods and [Strømhaug et al., 1998]). (B) Immunoblot of cell membrane fractions from GFP-LC3B expressing WT HEK293 cells showing enrichment of ATG9A and LC3B together in fractions 5 and 6, fraction 6 being reportedly enriched for autophagosome membranes. Cells were grown in DMEM before being starved in EBSS and treated with 0.1 µM bafilomycin A1 for 4–6 h. Loaded protein: 3 µg. (C) Immunoblot of cell membrane fractions from GFP-LC3B expressing ATG2 DKO HEK293 cells showing enrichment of ATG9A and LC3B together in fraction 5, while fraction 6 is largely depleted of both proteins in this cell line blocking autophagosome formation. Cells were grown in DMEM under basal conditions (ATG2 DKO cells accumulate early autophagic factors without starvation or bafilomycin A1 treatment [Valverde et al., 2019]). Loaded protein: 3 µg. (D) Densitometric quantification of ATG9A, GFP-LC3B, LC3B-II, Calnexin, and TOM20 in each cell line membrane fractionation displayed as average ± SD. The intensity of the bands in B and C 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, n = 4 biological replicates; ATG2 DKO, n = 3 biological replicates. (E) Densitometric quantification of ATG9A in each fraction compared between the two cells lines and with WT cells grown under basal conditions displayed as average ± SD. The intensity of the bands in B and C were normalized to the lysate, and statistical significance was assessed by two-way ANOVA. *, adjusted P value <0.05. ***, adjusted P value <0.001. WT Basal, n = 3 biological replicates; WT Starved+Baf, n = 4 biological replicates; ATG2 DKO, n = 3 biological replicates. More quantifications are shown in Fig. S5. Basal WT membrane fractions are shown in Fig. S4. Baf, bafilomycin A1.](https://cdn.rupress.org/rup/content_public/journal/jcb/222/7/10.1083_jcb.202208088/2/jcb_202208088_fig3.png?Expires=1771653483&Signature=Pympbg6qxSOUtfDNrVyEZP9SucByzMAHcmje2Zm7rj4lv97nb2-e2AIWylepHC8QdaRQ-WlQVwZKRjY7eRL33fD41ejbQGbNOfXcVqWTvtBRPmtLZ3CC75goFIKnn8njoNCaiNocDxt95LNgbHpWl7Ap~kGFCesxnM9Z75lWNiBB4ERZ511ujZmxNkdrxcXvwozwV~J5JH7GJzwKAdvLS5sQVLZfTvu6zptFD-69la1onNTKU5zHYqlMm1W4FP~KjvSL0bDYFp6A0~sBQvKhSxjYlGt1Y1THgQ43bTbNCSCCeRc~QGLVnw2W2u-zAQXl725Q1t8tPzhGM9jqQQEl8g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
ATG9A and LC3B reside in the same membrane fractions enriched for autophagosomes or their precursor membranes. (A) Experimental scheme for density gradient membrane fractionation protocol (see Materials and methods and [Strømhaug et al., 1998]). (B) Immunoblot of cell membrane fractions from GFP-LC3B expressing WT HEK293 cells showing enrichment of ATG9A and LC3B together in fractions 5 and 6, fraction 6 being reportedly enriched for autophagosome membranes. Cells were grown in DMEM before being starved in EBSS and treated with 0.1 µM bafilomycin A1 for 4–6 h. Loaded protein: 3 µg. (C) Immunoblot of cell membrane fractions from GFP-LC3B expressing ATG2 DKO HEK293 cells showing enrichment of ATG9A and LC3B together in fraction 5, while fraction 6 is largely depleted of both proteins in this cell line blocking autophagosome formation. Cells were grown in DMEM under basal conditions (ATG2 DKO cells accumulate early autophagic factors without starvation or bafilomycin A1 treatment [Valverde et al., 2019]). Loaded protein: 3 µg. (D) Densitometric quantification of ATG9A, GFP-LC3B, LC3B-II, Calnexin, and TOM20 in each cell line membrane fractionation displayed as average ± SD. The intensity of the bands in B and C 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, n = 4 biological replicates; ATG2 DKO, n = 3 biological replicates. (E) Densitometric quantification of ATG9A in each fraction compared between the two cells lines and with WT cells grown under basal conditions displayed as average ± SD. The intensity of the bands in B and C were normalized to the lysate, and statistical significance was assessed by two-way ANOVA. *, adjusted P value <0.05. ***, adjusted P value <0.001. WT Basal, n = 3 biological replicates; WT Starved+Baf, n = 4 biological replicates; ATG2 DKO, n = 3 biological replicates. More quantifications are shown in Fig. S5. Basal WT membrane fractions are shown in Fig. S4. Baf, bafilomycin A1.
