Figure 4. Rab5c activation by EGF stimuli facilitates AMAP1–PRKD2 interaction. (A) GST-AMAP1 and Xpress-Rab5c were overexpressed in 293T cells. GST-AMAP1 was precipitated by glutathione beads, and coprecipitation of Xpress-Rab5c was analyzed by immunoblotting. GST alone was also used as a control. (B and C) MDA-MB-231 cells cultured on collagen I–coated dishes were serum-starved for 2 h and stimulated with 10 ng/ml EGF (5 min). The GTP-bound form of endogenous Rab5c was precipitated by GST-R5BD (B). Endogenous Rab5c was immunoprecipitated, and coprecipitation of AMAP1 was analyzed by immunoblotting. Nonimmune rabbit IgG was used as a control (Non-imm). (D) GST-AMAP1, immobilized on glutathione beads, was incubated with either Xpress-tagged Rab5c-Q86L or -S35N. Coprecipitation of Rab5c mutants was analyzed by immunoblotting. (E) MDA-MB-231 cells were transfected with Arf6 or Rab5c siRNAs or a dsRNA with a nontargeting, irrelevant sequence (Irr), and cultured on collagen I-coated dishes. Cells were serum-starved for 2 h and treated (+) or untreated (−) with 10 ng/ml EGF (5 min). Cells were lysed and AMAP1 was immunoprecipitated. Co-precipitation of PRKD2 was analyzed by immunoblotting. (F and G) MDA-MB-231 cells were transfected with Rab5c siRNA or a dsRNA with a nontargeting, irrelevant sequence (Irr). Total lysates were subjected to blotting with the indicated antibodies (F). Matrigel chemoinvasion activities in the presence or absence of 10 ng/ml EGF are shown (G). Data are presented as percentages calculated by normalizing the values obtained for the irrelevant dsRNA-treated, EGF-untreated control cells as 100%. 732 ± 71 (∼0.73%) control cells were calculated to have transmigrated per 6.4-mm-diam Matrigel-coated Boyden chamber filter under this condition. Data are shown as mean ± SEM of triplicate experiments (error bars).
Figure 4.

Rab5c activation by EGF stimuli facilitates AMAP1–PRKD2 interaction. (A) GST-AMAP1 and Xpress-Rab5c were overexpressed in 293T cells. GST-AMAP1 was precipitated by glutathione beads, and coprecipitation of Xpress-Rab5c was analyzed by immunoblotting. GST alone was also used as a control. (B and C) MDA-MB-231 cells cultured on collagen I–coated dishes were serum-starved for 2 h and stimulated with 10 ng/ml EGF (5 min). The GTP-bound form of endogenous Rab5c was precipitated by GST-R5BD (B). Endogenous Rab5c was immunoprecipitated, and coprecipitation of AMAP1 was analyzed by immunoblotting. Nonimmune rabbit IgG was used as a control (Non-imm). (D) GST-AMAP1, immobilized on glutathione beads, was incubated with either Xpress-tagged Rab5c-Q86L or -S35N. Coprecipitation of Rab5c mutants was analyzed by immunoblotting. (E) MDA-MB-231 cells were transfected with Arf6 or Rab5c siRNAs or a dsRNA with a nontargeting, irrelevant sequence (Irr), and cultured on collagen I-coated dishes. Cells were serum-starved for 2 h and treated (+) or untreated (−) with 10 ng/ml EGF (5 min). Cells were lysed and AMAP1 was immunoprecipitated. Co-precipitation of PRKD2 was analyzed by immunoblotting. (F and G) MDA-MB-231 cells were transfected with Rab5c siRNA or a dsRNA with a nontargeting, irrelevant sequence (Irr). Total lysates were subjected to blotting with the indicated antibodies (F). Matrigel chemoinvasion activities in the presence or absence of 10 ng/ml EGF are shown (G). Data are presented as percentages calculated by normalizing the values obtained for the irrelevant dsRNA-treated, EGF-untreated control cells as 100%. 732 ± 71 (∼0.73%) control cells were calculated to have transmigrated per 6.4-mm-diam Matrigel-coated Boyden chamber filter under this condition. Data are shown as mean ± SEM of triplicate experiments (error bars).

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