Figure 2.
TMEM163 is localized to perinuclear DG and LE marker–positive compartments (likely DG precursors) and small vesicular structures (likely EEs) in the periphery, but not to AGs in MEG-01 cells. (A-B) MEG-01 cells were cotransfected with TMEM163-GFP or Cherry-TMEM163 and Cherry-VMAT2 (A) or GFP-VMAT2 (B) for 48 hours, respectively. (C) MEG-01 cells were transfected with Cherry-TMEM163 for 48 hours and then treated with 50 μM mepacrine for 30 minutes at 37°C. (D) MEG-01 cells were transfected with TMEM163-GFP for 48 hours and then fixed and stained with antibodies against CD63. (E-F) MEG-01 cells were cotransfected with Cherry-TMEM163 and GFP-Rab7 (E) or GFP-Lamp1 (F) for 48 hours, respectively. (G) MEG-01 cells were cotransfected with GFP-TMEM163 and Cherry-TPCN2 for 48 hours, respectively. (H) MEG-01 cells were transfected with Cherry-TMEM163 for 48 hours and treated with LysoSensor Green DND-189 for 30 minutes at 37°C. (I) MEG-01 cells were transfected with TMEM163-GFP for 48 hours and then fixed and stained with antibodies against EEA1. (J) MEG-01 cells were cotransfected with Cherry-TMEM163 and GFP-Rab5 for 48 hours. (K-L) MEG-01 cells were transfected with GFP-TMEM163 for 48 hours and then fixed and stained with antibodies against P-selectin (K) or VWF (L). Pictures shown in panels A-L are representative confocal images. Outlines of cells are indicated by white lines. Insets show 5× magnified images of the boxed region. Scale bars, 10 μm. (M) Colocalization analysis of results shown in panels A-L; PCC, 0.74 ± 0.04, 0.76 ± 0.02, 0.68 ± 0.04, 0.73 ± 0.03, 0.72 ± 0.03, 0.75 ± 0.02, 0.59 ± 0.04, 0.82 ± 0.02, 0.40 ± 0.02, 0.63 ± 0.03, 0.13 ± 0.02, and 0.04 ± 0.01; n = 11, 14, 8, 17, 7, 14, 13, 15, 22, 19, 10, and 10 cells, respectively. PCC ≥0.4 (dotted line) represents colocalization. (N) Sucrose (10% to 60%) gradient assay shows that TMEM163 coexists with the EE marker EEA1, the LE marker Rab7, and the DG marker LAMP2. Endo, endogenous; IB, immunoblotting.

TMEM163 is localized to perinuclear DG and LE marker–positive compartments (likely DG precursors) and small vesicular structures (likely EEs) in the periphery, but not to AGs in MEG-01 cells. (A-B) MEG-01 cells were cotransfected with TMEM163-GFP or Cherry-TMEM163 and Cherry-VMAT2 (A) or GFP-VMAT2 (B) for 48 hours, respectively. (C) MEG-01 cells were transfected with Cherry-TMEM163 for 48 hours and then treated with 50 μM mepacrine for 30 minutes at 37°C. (D) MEG-01 cells were transfected with TMEM163-GFP for 48 hours and then fixed and stained with antibodies against CD63. (E-F) MEG-01 cells were cotransfected with Cherry-TMEM163 and GFP-Rab7 (E) or GFP-Lamp1 (F) for 48 hours, respectively. (G) MEG-01 cells were cotransfected with GFP-TMEM163 and Cherry-TPCN2 for 48 hours, respectively. (H) MEG-01 cells were transfected with Cherry-TMEM163 for 48 hours and treated with LysoSensor Green DND-189 for 30 minutes at 37°C. (I) MEG-01 cells were transfected with TMEM163-GFP for 48 hours and then fixed and stained with antibodies against EEA1. (J) MEG-01 cells were cotransfected with Cherry-TMEM163 and GFP-Rab5 for 48 hours. (K-L) MEG-01 cells were transfected with GFP-TMEM163 for 48 hours and then fixed and stained with antibodies against P-selectin (K) or VWF (L). Pictures shown in panels A-L are representative confocal images. Outlines of cells are indicated by white lines. Insets show 5× magnified images of the boxed region. Scale bars, 10 μm. (M) Colocalization analysis of results shown in panels A-L; PCC, 0.74 ± 0.04, 0.76 ± 0.02, 0.68 ± 0.04, 0.73 ± 0.03, 0.72 ± 0.03, 0.75 ± 0.02, 0.59 ± 0.04, 0.82 ± 0.02, 0.40 ± 0.02, 0.63 ± 0.03, 0.13 ± 0.02, and 0.04 ± 0.01; n = 11, 14, 8, 17, 7, 14, 13, 15, 22, 19, 10, and 10 cells, respectively. PCC ≥0.4 (dotted line) represents colocalization. (N) Sucrose (10% to 60%) gradient assay shows that TMEM163 coexists with the EE marker EEA1, the LE marker Rab7, and the DG marker LAMP2. Endo, endogenous; IB, immunoblotting.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal