Figure 5. Rab3GAP1/2 controls the activity and LD localization of Rab18. (A) Endogenous Rab18 (green) was associated with LDs (red) in 3T3-L1 preadipocytes. Bars: 10 µm; (insets) 2 µm. (B) Rab18 was enriched in LD fraction isolated from 3T3-L1 preadipocytes. ADRP, LD marker; GRP94, a microsomal marker; β-tubulin, a cytosol marker. (C) Rab18 (green) was associated with LD (blue) at early stage of LD biogenesis. Red, endogenous ACSL3. Bars: 10 µm; (insets) 2 µm. (D) Representative SIM superresolution images showing the localization of HA-Rab3GAP2 (red) and GFP-Rab18 (green) on LDs (blue). Bars: 10 µm; (insets) 1 µm. (E) Rab18 (green) was not localized to LDs (blue) in Rab3GAP1- or Rab3GAP2-deficient cells. Red, ACSL3. Bars: 10 µm; (insets) 2 µm. (F) Rab18 was not enriched in the LD fraction in Rab3GAP1-deficient cells. (G and H) Reduced mature LDs and the presence of supersized LDs in Rab3GAP1/2-deficient cells. Bars: 10 µm; (insets) 2 µm. Histogram in H showing the mean number of LDs in each diameter in G. Mean ± SD; n = 13 for control cells, n = 20 for Rab3GAP1 KO cells, n = 16 for Rab3GAP2 KO cells; ***, P < 0.001 by one-way ANOVA with Tukey post hoc tests. (I) Increased LD (green) sizes in Rab3GAP1-deficient adipocytes. Bars: 10 µm; (insets) 5 µm. All experiments were performed at least twice.
Figure 5.

Rab3GAP1/2 controls the activity and LD localization of Rab18. (A) Endogenous Rab18 (green) was associated with LDs (red) in 3T3-L1 preadipocytes. Bars: 10 µm; (insets) 2 µm. (B) Rab18 was enriched in LD fraction isolated from 3T3-L1 preadipocytes. ADRP, LD marker; GRP94, a microsomal marker; β-tubulin, a cytosol marker. (C) Rab18 (green) was associated with LD (blue) at early stage of LD biogenesis. Red, endogenous ACSL3. Bars: 10 µm; (insets) 2 µm. (D) Representative SIM superresolution images showing the localization of HA-Rab3GAP2 (red) and GFP-Rab18 (green) on LDs (blue). Bars: 10 µm; (insets) 1 µm. (E) Rab18 (green) was not localized to LDs (blue) in Rab3GAP1- or Rab3GAP2-deficient cells. Red, ACSL3. Bars: 10 µm; (insets) 2 µm. (F) Rab18 was not enriched in the LD fraction in Rab3GAP1-deficient cells. (G and H) Reduced mature LDs and the presence of supersized LDs in Rab3GAP1/2-deficient cells. Bars: 10 µm; (insets) 2 µm. Histogram in H showing the mean number of LDs in each diameter in G. Mean ± SD; n = 13 for control cells, n = 20 for Rab3GAP1 KO cells, n = 16 for Rab3GAP2 KO cells; ***, P < 0.001 by one-way ANOVA with Tukey post hoc tests. (I) Increased LD (green) sizes in Rab3GAP1-deficient adipocytes. Bars: 10 µm; (insets) 5 µm. All experiments were performed at least twice.

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