Figure 7. HMMR is a microtubule-binding protein and partner of CHICA. (A) HeLa cells were transfected with full-length GFP-tagged HMMR or the deletion constructs outlined in the schematic. After 24 h the cells were fixed and then stained for tubulin, and DAPI to detect DNA. HMMR was visualized using GFP fluorescence. Representative examples of the localization in mitotic or interphase cells are shown. Bar, 10 µm. (B) Microtubule-binding assays were performed using in vitro–translated full-length HMMR and the fragments shown in the figure. The anaphase spindle protein PRC1 and the small GTPase Rab4 were taken as positive and negative controls, respectively. (C) HEK293T cells were cotransfected with full-length and deletion constructs of GFP-HMMR and Myc-CHICA for 30 h. HMMR complexes were immunoprecipitated using sheep anti-GFP antibodies, and then Western blotted using mouse anti-GFP or mouse anti-Myc. An asterisk marks a nonspecific band in the Myc blots. (D) HeLa cells were transfected with full-length GFP-tagged CHICA or the deletion constructs outlined in the schematic. After 24 h, the cells were fixed and then stained for tubulin, and DAPI to detect DNA. CHICA was visualized using GFP fluorescence. Representative examples of the localization in mitotic or interphase cells are shown. Bar, 10 µm. (E) Microtubule-binding assays were performed using in vitro–translated full-length CHICA and the fragments shown in the figure. The anaphase spindle protein PRC1 and the small GTPase Rab4 were taken as positive and negative controls, respectively. (F) HEK293T cells were cotransfected with full-length and deletion constructs of GFP-CHICA and Myc-HMMR for 30 h. CHICA complexes were immunoprecipitated using sheep anti-GFP antibodies, and then Western blotted using mouse anti-GFP or mouse anti-Myc.
Figure 7.

HMMR is a microtubule-binding protein and partner of CHICA. (A) HeLa cells were transfected with full-length GFP-tagged HMMR or the deletion constructs outlined in the schematic. After 24 h the cells were fixed and then stained for tubulin, and DAPI to detect DNA. HMMR was visualized using GFP fluorescence. Representative examples of the localization in mitotic or interphase cells are shown. Bar, 10 µm. (B) Microtubule-binding assays were performed using in vitro–translated full-length HMMR and the fragments shown in the figure. The anaphase spindle protein PRC1 and the small GTPase Rab4 were taken as positive and negative controls, respectively. (C) HEK293T cells were cotransfected with full-length and deletion constructs of GFP-HMMR and Myc-CHICA for 30 h. HMMR complexes were immunoprecipitated using sheep anti-GFP antibodies, and then Western blotted using mouse anti-GFP or mouse anti-Myc. An asterisk marks a nonspecific band in the Myc blots. (D) HeLa cells were transfected with full-length GFP-tagged CHICA or the deletion constructs outlined in the schematic. After 24 h, the cells were fixed and then stained for tubulin, and DAPI to detect DNA. CHICA was visualized using GFP fluorescence. Representative examples of the localization in mitotic or interphase cells are shown. Bar, 10 µm. (E) Microtubule-binding assays were performed using in vitro–translated full-length CHICA and the fragments shown in the figure. The anaphase spindle protein PRC1 and the small GTPase Rab4 were taken as positive and negative controls, respectively. (F) HEK293T cells were cotransfected with full-length and deletion constructs of GFP-CHICA and Myc-HMMR for 30 h. CHICA complexes were immunoprecipitated using sheep anti-GFP antibodies, and then Western blotted using mouse anti-GFP or mouse anti-Myc.

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