Figure 3. Proteasomal degradation of... | Rockefeller University Press

Figure 3.

$Proteasomal degradation of newly synthesized caveolins. (A–C) The indicated proteins were expressed (as in Fig. 2 A) in control (CTL) or in cells additionally treated with MG132 (MG). Protein steady-state levels determined after 3 h by IB with anti-GFP antibodies (A; as in Fig. 2 B). Proteins levels were referred to the expression of each protein in the absence of MG132 (red line; B; n ≥ 4 independent experiments). CAV1 K*R and CAV3 K*R steady-state levels were compared to CTL and MG132-treated cells and referred to the wt caveolin (C; n ≥ 3 independent experiments). (D–G) CAV1 K*R and CAV3 K*R were expressed for 3 h, and their intracellular distribution was analyzed by confocal microscopy. D and E show representative images of the mutants and their co-localization with Calreticulin (ER marker). Arrows indicate punctate structures formed by the mutants and are especially apparent along the ER. Additional images are included in Fig. S3. F and G show the colocalization of the mutants with markers of each compartment in confocal microscopy images quantified using the Manders M2 overlapping coefficient (n ≥ 3 independent experiments and at least nine cells per condition). (H–O) The indicated proteins were expressed in CTL cells or, in the case of CAV1 and CAV3, in cells additionally treated with MG. After 3 h, cells were solubilized with either TX or TX+SDS and fractionated in sucrose sedimentation gradients (as in Fig. 2, K and M). Protein distribution was determined in equal volumes of each fraction by IB with anti-GFP antibodies. H and K show a representative result and (I–O) the relative distribution of the proteins in each fraction of the gradients (n = 3 independent experiments). All graphs show means ± SD; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 calculated in a one-way ANOVA test (B and C) and two-way ANOVA test (F, G, I, J, L, and O). Scale bars are 20 μm. Source data are available for this figure: SourceData F3.$

Proteasomal degradation of newly synthesized caveolins. (A–C) The indicated proteins were expressed (as in Fig. 2 A) in control (CTL) or in cells additionally treated with MG132 (MG). Protein steady-state levels determined after 3 h by IB with anti-GFP antibodies (A; as in Fig. 2 B). Proteins levels were referred to the expression of each protein in the absence of MG132 (red line; B; n ≥ 4 independent experiments). CAV1 K*R and CAV3 K*R steady-state levels were compared to CTL and MG132-treated cells and referred to the wt caveolin (C; n ≥ 3 independent experiments). (D–G) CAV1 K*R and CAV3 K*R were expressed for 3 h, and their intracellular distribution was analyzed by confocal microscopy. D and E show representative images of the mutants and their co-localization with Calreticulin (ER marker). Arrows indicate punctate structures formed by the mutants and are especially apparent along the ER. Additional images are included in Fig. S3. F and G show the colocalization of the mutants with markers of each compartment in confocal microscopy images quantified using the Manders M2 overlapping coefficient (n ≥ 3 independent experiments and at least nine cells per condition). (H–O) The indicated proteins were expressed in CTL cells or, in the case of CAV1 and CAV3, in cells additionally treated with MG. After 3 h, cells were solubilized with either TX or TX+SDS and fractionated in sucrose sedimentation gradients (as in Fig. 2, K and M). Protein distribution was determined in equal volumes of each fraction by IB with anti-GFP antibodies. H and K show a representative result and (I–O) the relative distribution of the proteins in each fraction of the gradients (n = 3 independent experiments). All graphs show means ± SD; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 calculated in a one-way ANOVA test (B and C) and two-way ANOVA test (F, G, I, J, L, and O). Scale bars are 20 μm. Source data are available for this figure: SourceData F3.

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