Figure 9. Overexpression of the wild-type and active form Rab33B inhibited fusion between autophagosomes and lysosomes. (A) Wild-type Rab33B and the active form Rab33B mutant (Rab33B-QL) increased the amount of LC3-II. Cell lysates from MEF cells stably expressing FLAG-Rab33B, FLAG–Rab33B-QL, or neither (control) were cultured under nutrient-rich (N), starved (S), or replenished (R) conditions, and their lysates were analyzed by immunoblotting with anti-OATL1 antibody (top), antiactin antibody (second panel), anti-FLAG tag antibody (third panel), and anti-LC3 antibody (bottom). (B) Expression of the wild-type and active form Rab33B increased the number of LC3-positive dots. MEF cells expressing FLAG-Rab33B, FLAG–Rab33B-QL, or neither (control) were cultured under the same conditions as in A. The cells cultured under each condition were fixed and stained with anti-LC3 antibody, and the numbers of LC3-positive dots per cell were counted (n ≥ 100). Error bars represent the means ± SEM of representative data (n ≥ 100) from two independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; one-way analysis of variance and Tukey posthoc test (compared with the control under the same conditions). (C) Overexpression of Rab33B-QL induced LC3-positive dots that were closely associated with Lamp-1–positive dots. MEF cells stably expressing FLAG–Rab33B-QL were cultured under replenished conditions, then fixed, and stained with anti-LC3 antibody (red) and anti–Lamp-1 antibody (green). Merged images are shown on the bottom. Higher magnification views of the boxed areas are shown at the right. Bar, 10 µm.
Figure 9.

Overexpression of the wild-type and active form Rab33B inhibited fusion between autophagosomes and lysosomes. (A) Wild-type Rab33B and the active form Rab33B mutant (Rab33B-QL) increased the amount of LC3-II. Cell lysates from MEF cells stably expressing FLAG-Rab33B, FLAG–Rab33B-QL, or neither (control) were cultured under nutrient-rich (N), starved (S), or replenished (R) conditions, and their lysates were analyzed by immunoblotting with anti-OATL1 antibody (top), antiactin antibody (second panel), anti-FLAG tag antibody (third panel), and anti-LC3 antibody (bottom). (B) Expression of the wild-type and active form Rab33B increased the number of LC3-positive dots. MEF cells expressing FLAG-Rab33B, FLAG–Rab33B-QL, or neither (control) were cultured under the same conditions as in A. The cells cultured under each condition were fixed and stained with anti-LC3 antibody, and the numbers of LC3-positive dots per cell were counted (n ≥ 100). Error bars represent the means ± SEM of representative data (n ≥ 100) from two independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; one-way analysis of variance and Tukey posthoc test (compared with the control under the same conditions). (C) Overexpression of Rab33B-QL induced LC3-positive dots that were closely associated with Lamp-1–positive dots. MEF cells stably expressing FLAG–Rab33B-QL were cultured under replenished conditions, then fixed, and stained with anti-LC3 antibody (red) and anti–Lamp-1 antibody (green). Merged images are shown on the bottom. Higher magnification views of the boxed areas are shown at the right. Bar, 10 µm.

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