Figure 4.
CBP is required for optimal ER-phagy under ER-stress conditions. (A–C) CBP-mediated acetylation for FAM134B is important for liposome fragmentation. Purified recombinant FAM134B protein was acetylated by CBP in vitro, followed by liposome fragmentation assay. After injecting recombinant proteins (25 μg) into the chamber (500 μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated were presented. The time required for liposome fragmentation was quantified in C (n = 3 experimental replicates). Scale bars, 10 μm. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (D and E) Overexpression of CBP enhanced FAM134B-mediated ER membrane fragmentation. FAM134B KO U2OS cells were engineered to inducibly express EGFP-FAM134B (WT) at endogenous levels. Cells were treated with Tg (1 μM, 1 h) or DMSO. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in E (n = 25 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001, ns means no significance. (F and G) ER membrane protein degradation analysis in CBP WT or KD cells. Cells were treated with Tg (1 μM) for 0, 0.5, 1, 3, 6 h. The indicated proteins were detected by Western blot. Quantification of K160 acetylation was shown in G (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. *P < 0.05, **P < 0.01. (H and I) Lysosomal cleavage of GFP was analyzed by Western blot in CBP WT or KD cells transfected with 1.5 μg GFP-SEC61B and 0.5 μg FAM134B-Flag. Cells were treated with Tg (1 μM) for 1 h or DMSO. Quantification of cleavaged GFP is shown in I (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01. (J and K) CBP KD impaired FAM134B-mediated ER-phagy. CBP WT or KD U2OS cells transiently expressing 0.2 µg mCherry-EGFP-FAM134B (WT). MCherry-positive but EGFP-negative puncta were quantified for in K (n = 30 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001, ns means no significance. (L) Acetylation of FAM134B-K160 by Tg treatment promotes the degradation of ER resident protein ATZ. HEK293T cells transfected with HA-ATZ were treated with Tg (1 µM), C646 (10 µM), or BafA1 (10 nM) for 6 h. The indicated proteins were detected by Western blot. The experiments were performed twice. (M) Acetylation of FAM134B-K160 by Tg treatment promotes the degradation of collagen I. Mouse embryonic fibroblast cells were treated with Tg (1 µM), C646 (10 µM) or BafA1 (10 nM) for 6 h. The indicated proteins were detected by Western blot. The experiments were performed twice. (N) Acetylation of FAM134B-K160 by Tg treatment promotes the degradation of ER resident protein NPC1 mutant (I1061T). HEK293T cells transfected with HA-NPC1(I1061T) were treated with Tg (1 µM), C646 (10 µM), or BafA1 (10 nM) for 6 h. The indicated proteins were detected by Western blot. The experiments were performed twice. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F4.

CBP is required for optimal ER-phagy under ER-stress conditions. (A–C) CBP-mediated acetylation for FAM134B is important for liposome fragmentation. Purified recombinant FAM134B protein was acetylated by CBP in vitro, followed by liposome fragmentation assay. After injecting recombinant proteins (25 μg) into the chamber (500 μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated were presented. The time required for liposome fragmentation was quantified in C (n = 3 experimental replicates). Scale bars, 10 μm. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (D and E) Overexpression of CBP enhanced FAM134B-mediated ER membrane fragmentation. FAM134B KO U2OS cells were engineered to inducibly express EGFP-FAM134B (WT) at endogenous levels. Cells were treated with Tg (1 μM, 1 h) or DMSO. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in E (n = 25 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001, ns means no significance. (F and G) ER membrane protein degradation analysis in CBP WT or KD cells. Cells were treated with Tg (1 μM) for 0, 0.5, 1, 3, 6 h. The indicated proteins were detected by Western blot. Quantification of K160 acetylation was shown in G (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. *P < 0.05, **P < 0.01. (H and I) Lysosomal cleavage of GFP was analyzed by Western blot in CBP WT or KD cells transfected with 1.5 μg GFP-SEC61B and 0.5 μg FAM134B-Flag. Cells were treated with Tg (1 μM) for 1 h or DMSO. Quantification of cleavaged GFP is shown in I (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01. (J and K) CBP KD impaired FAM134B-mediated ER-phagy. CBP WT or KD U2OS cells transiently expressing 0.2 µg mCherry-EGFP-FAM134B (WT). MCherry-positive but EGFP-negative puncta were quantified for in K (n = 30 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001, ns means no significance. (L) Acetylation of FAM134B-K160 by Tg treatment promotes the degradation of ER resident protein ATZ. HEK293T cells transfected with HA-ATZ were treated with Tg (1 µM), C646 (10 µM), or BafA1 (10 nM) for 6 h. The indicated proteins were detected by Western blot. The experiments were performed twice. (M) Acetylation of FAM134B-K160 by Tg treatment promotes the degradation of collagen I. Mouse embryonic fibroblast cells were treated with Tg (1 µM), C646 (10 µM) or BafA1 (10 nM) for 6 h. The indicated proteins were detected by Western blot. The experiments were performed twice. (N) Acetylation of FAM134B-K160 by Tg treatment promotes the degradation of ER resident protein NPC1 mutant (I1061T). HEK293T cells transfected with HA-NPC1(I1061T) were treated with Tg (1 µM), C646 (10 µM), or BafA1 (10 nM) for 6 h. The indicated proteins were detected by Western blot. The experiments were performed twice. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F4.

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