Figure 7.
GRAF/WDR44 label a subset of tubular endosomes. (A–C) HeLa cells were incubated with methanol (vehicle) or BFA (5 µg/ml) for 5 or 15 min. (A) Percentage of cells with endogenous (endo.) WDR44 tubules. n = 7–25. (B) Confocal images of cells stained with α-WDR44 and α-TGN46 or preincubated with Alexa Fluor 546–Transferrin (10 µg/ml, 1 h). (C) Manders colocalization coefficients for the indicated proteins with endogenous WDR44 structures. n = 10–50 cells. (D) Confocal images of transfected HeLa cells showing colocalization of RFP-WDR44 with GFP-STX16 and GFP-VAMP3. (E) Immunoprecipitation (IP) of transfected 293T cells with α-GFP. uGRAF2 was coimmunoprecipitated by GFP-MICAL1, but not by any other member of the MICAL family. (F) Confocal images of transfected HeLa cells stained with α-WDR44. In the case of MICAL3pF1KA0819 and MICAL-L2, boxed areas show tubules positive only for WDR44 (red), only for MICAL3pF1KA0819/MICAL-L2 (cyan), or shared by the two proteins (white). (G) Percentage of transfected HeLa cells with endogenous (endo.) WDR44 tubules. n = 4. (H) Confocal images of untransfected HeLa cells stained with α-WDR44 and α-MICAL-L1. Boxed areas show tubules positive only for WDR44 (red), only for MICAL-L1 (cyan), or shared by the two proteins (white). (I) Internalized Integrin-β1 in shRNA-expressing HeLa cells after uptake of α-Integrin-β1 and following a 4-h chase. n = 4. (A, C, G, and I) Data are means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. (B, D, F, and H) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

GRAF/WDR44 label a subset of tubular endosomes. (A–C) HeLa cells were incubated with methanol (vehicle) or BFA (5 µg/ml) for 5 or 15 min. (A) Percentage of cells with endogenous (endo.) WDR44 tubules. n = 7–25. (B) Confocal images of cells stained with α-WDR44 and α-TGN46 or preincubated with Alexa Fluor 546–Transferrin (10 µg/ml, 1 h). (C) Manders colocalization coefficients for the indicated proteins with endogenous WDR44 structures. n = 10–50 cells. (D) Confocal images of transfected HeLa cells showing colocalization of RFP-WDR44 with GFP-STX16 and GFP-VAMP3. (E) Immunoprecipitation (IP) of transfected 293T cells with α-GFP. uGRAF2 was coimmunoprecipitated by GFP-MICAL1, but not by any other member of the MICAL family. (F) Confocal images of transfected HeLa cells stained with α-WDR44. In the case of MICAL3pF1KA0819 and MICAL-L2, boxed areas show tubules positive only for WDR44 (red), only for MICAL3pF1KA0819/MICAL-L2 (cyan), or shared by the two proteins (white). (G) Percentage of transfected HeLa cells with endogenous (endo.) WDR44 tubules. n = 4. (H) Confocal images of untransfected HeLa cells stained with α-WDR44 and α-MICAL-L1. Boxed areas show tubules positive only for WDR44 (red), only for MICAL-L1 (cyan), or shared by the two proteins (white). (I) Internalized Integrin-β1 in shRNA-expressing HeLa cells after uptake of α-Integrin-β1 and following a 4-h chase. n = 4. (A, C, G, and I) Data are means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. (B, D, F, and H) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

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