Fig 1: MYTHO interacts with autophagic proteins.(A) Mass spectrometry analysis of immunoprecipitated endogenous HA-tagged MYTHO. Significant autophagy-related proteins are shown in the graph. (B) Quantification of WIPI2 puncta in Fed and starved (2 hours with Earle’s balanced salt solution [EBSS]) conditions in WT and Mytho-KO C2C12 cells in N = 3. Fed WT (n = 141); Fed Mytho-KO (n = 144), EBSS 2 h WT (n = 139); EBSS 2 h Mytho-KO (n = 172). Bars indicate SEM. ****P < 0.0001 (1-way ANOVA with Tukey’s multiple-comparison test). (C) HEK293 cells transfected with WIPI2-GFP or GFP were immunoprecipitated with GFP-TRAP. The quantification of N = 3 (normalized by input) is shown in Supplemental Figure 9E. (D) HEK293 cells transfected with MYTHO-GFP or GFP were immunoprecipitated. Endogenous WIPI2, ATG7, and BCAS3 were immunoblotted. In the blot on the left, lanes were run on the same gel but were noncontiguous. The quantification of N = 3 (normalized by input) is represented in Supplemental Figure 9F. N = number of independent experiments; n = number of cells/samples.
Fig 2: MYTHO is required for WIPI2 and BCAS3 localization on autophagosomes.(A) Representative scheme showing predicted LC3 interaction motifs and WD40 domains: Y91A/V94A (M1); F131A/L134A (M2); Y288A/L291A (M3); W351A/I354A (M4); 208delTGPSGLRLRL (M5) or Y288A/L291A + 208delTGPSGLRLRL (M3/M5). (B and C) HEK293 cells transfected with GFP, MYTHO-GFP, or MYTHO-GFP mutants were lysed and immunoprecipitated with GFP-TRAP, and blotted with indicated markers. All samples were run on the same gel. Quantification of LC3, WIPI2, and BCAS3 enrichment (normalized to input) is shown in Supplemental Figure 10, B–D (N = 3). (D) HEK293 cells were transfected with the following vectors: empty (GFP), GFP-WIPI2 or WIPI2 mutants (GFP-RERE [R108E/R128E], GFP-FTTG, or double mutant). Immunoprecipitation was performed as in B and C, and endogenous BCAS3 or MYTHO was blotted. (E) Left: Representative fluorescence images of endogenous WIPI2 protein in Mytho-KO cells transfected with GFP or MYTHO-GFP vector. Scale bars: 20 μm. Right: Quantification of WIPI2 puncta/cell in Fed and 2-hour starved (STV) (N = 3) using ImageJ software. Fed WT + GFP (n = 34); Fed Mytho-KO + GFP (n = 69), Fed Mytho-KO + MYTHO-GFP (n = 49); HBSS 2h WT + GFP (n = 12); HBSS 2h Mytho-KO + GFP (n = 53), HBSS 2h Mytho-KO + MYTHO-GFP (n = 33) (1-way ANOVA on ranks [Kruskal-Wallis test]). (F and G) WT and Mytho-KO C2C12 cells were transfected with empty (GFP), MYTHO-GFP (WT), M1-GFP, M3-GFP, M5-GFP, or M3/M5-GFP vector. The quantification of endogenous LC3 (F) or WIPI2 (G) puncta in the fed condition was performed using ImageJ software (N = 3). For LC3 puncta: WT + GFP (n = 91); Mytho-KO + GFP (n = 101), Mytho-KO + WT (n = 84); Mytho-KO + M1 (n = 41); Mytho-KO + M3 (n = 48), Mytho-KO + M5 (n = 40), Mytho-KO + M3/M5 (n = 53) (1-way ANOVA with Tukey’s multiple-comparison test). For WIPI2 puncta: WT + GFP (n = 107); Mytho-KO + GFP (n = 119), Mytho-KO + WT (n = 98); Mytho-KO + M1 (n = 65); Mytho-KO + M3 (n = 115), Mytho-KO + M5 (n = 50), Mytho-KO + M3/M5 (n = 56) (1-way ANOVA on ranks [Kruskal-Wallis test]). All bars indicate SEM. **P < 0.001; ****P < 0.0001. N = number of independent experiments; n = number of samples.
Supplier Page from Abcam for Anti-BCAS3 antibody