Figure 4. Membrane association of Rab11b... | Rockefeller University Press

Figure 4.

$Figure 4. Membrane association of Rab11b is regulated by MVP, GGT-II, and mutant p53, and Rab11b is responsible for the PM recruitment of Arf6 in MDA-MB-231 cells. (A and B) Ligand-induced Matrigel invasion activities (A) and Arf6 activation (B) were measured in cells pretreated with the indicated siRNAs or Irr. Matrigel invasion activities were measured as described in the Matrigel invasion assay section within Materials and methods. n = 3. (C and D) Intracellular colocalization of Arf6-mCherry and EGFP-Rab11a (top) or Arf6-EGFP and RFP-Rab11b (bottom) was examined using high-resolution SIM (bars, 10 µm; C). Pearson’s correlation coefficients of the intracellular colocalization of these proteins, as indicated, were estimated from >10 cells as described in the Immunofluorescence microscopy section within Materials and methods. n = 2 (D). (E) Intracellular colocalization of endogenous Rab11b with Arf6-EGFP was examined using deconvolution microscopy. Enlarged views of the boxed areas are shown on the right. Bars: (right) 5 µm; (left) 10 µm. (F and G) The PM localization of Arf6-EGFP was examined in cells pretreated with siRNAs for Rab11a, Rab11b, Rab11a/11b, or Irr, before (−) and after (+) 5 min TGFβ1 stimulation. F-actin was visualized using Alexa Fluor 647–conjugated phalloidin (F). Relative amounts of Arf6-EGFP localized at the cell periphery (G) were estimated from >10 cells as described in the Immunofluorescence microscopy section. n = 2. (H–J) The membrane association of HA-Rab11b was measured in cells pretreated with or without Simvastatin for 24 h (H) or pretreated with siRNAs for GGT-I (siRNA #1) or GGT-II (siRNA #1; I) and in parental cells and their p53 derivatives (J). In H, the Rab11b C214A/C215A mutant was used as a control. wt, wild-type Rab11b. S, soluble fraction. M, membrane fraction. Representative images are shown from two independent experiments in which >10 cells were examined in each experiment. Bars, 10 µm. The results represent mean ± SEM. *, P < 0.001.$

Membrane association of Rab11b is regulated by MVP, GGT-II, and mutant p53, and Rab11b is responsible for the PM recruitment of Arf6 in MDA-MB-231 cells. (A and B) Ligand-induced Matrigel invasion activities (A) and Arf6 activation (B) were measured in cells pretreated with the indicated siRNAs or Irr. Matrigel invasion activities were measured as described in the Matrigel invasion assay section within Materials and methods. n = 3. (C and D) Intracellular colocalization of Arf6-mCherry and EGFP-Rab11a (top) or Arf6-EGFP and RFP-Rab11b (bottom) was examined using high-resolution SIM (bars, 10 µm; C). Pearson’s correlation coefficients of the intracellular colocalization of these proteins, as indicated, were estimated from >10 cells as described in the Immunofluorescence microscopy section within Materials and methods. n = 2 (D). (E) Intracellular colocalization of endogenous Rab11b with Arf6-EGFP was examined using deconvolution microscopy. Enlarged views of the boxed areas are shown on the right. Bars: (right) 5 µm; (left) 10 µm. (F and G) The PM localization of Arf6-EGFP was examined in cells pretreated with siRNAs for Rab11a, Rab11b, Rab11a/11b, or Irr, before (−) and after (+) 5 min TGFβ1 stimulation. F-actin was visualized using Alexa Fluor 647–conjugated phalloidin (F). Relative amounts of Arf6-EGFP localized at the cell periphery (G) were estimated from >10 cells as described in the Immunofluorescence microscopy section. n = 2. (H–J) The membrane association of HA-Rab11b was measured in cells pretreated with or without Simvastatin for 24 h (H) or pretreated with siRNAs for GGT-I (siRNA #1) or GGT-II (siRNA #1; I) and in parental cells and their p53 derivatives (J). In H, the Rab11b C214A/C215A mutant was used as a control. wt, wild-type Rab11b. S, soluble fraction. M, membrane fraction. Representative images are shown from two independent experiments in which >10 cells were examined in each experiment. Bars, 10 µm. The results represent mean ± SEM. *, P < 0.001.