Figure 2. Subcellular distribution... | Rockefeller University Press

Figure 2.

$Figure 2. Subcellular distribution analyses of CIMPR, TGN46, and TfnR in HeLa cells depleted of or overexpressing mDpy-30. All siRNAs were used at a concentration of 20 nM, and transfected cells were analyzed 48 h after transfection. Unless noted otherwise, the values shown in all bar graphs and Western blots are the mean results obtained from at least three independent experiments. For imaging analyses, >500 cells were counted in each experiment. (A) Subcellular distributions of CIMPR, TGN46, or TfnR in HeLa cells treated with control nontargeting or mDpy-30 siRNAs. Cells were processed as described in Fig. 1. Arrows indicate cell protrusions. (B) Enrichment of CIMPR near cell protrusions (indicated by arrows) was confirmed by phalloidin F-actin costaining, and the fraction of cells displaying enriched CIMPR near protrusions was scored. (C) Western blot analysis of the CIMPR protein level after the knockdown of mDpy-30. The numbers represent the mean value of six independent experiments. (D) The effect of mDpy-30 overexpression on the localization of CIMPR or β-GalT1 (compare asterisks with arrows). Only those cells exhibiting a high level of exogenous mDpy-30 were counted. The experiment was repeated once, and the mean value is shown. (E) The protein level of CIMPR in cells overexpressing mDpy-30. (B and E) Error bars indicate the standard deviation of the values obtained from independent experiments. Bars, 10 µm.$

Subcellular distribution analyses of CIMPR, TGN46, and TfnR in HeLa cells depleted of or overexpressing mDpy-30. All siRNAs were used at a concentration of 20 nM, and transfected cells were analyzed 48 h after transfection. Unless noted otherwise, the values shown in all bar graphs and Western blots are the mean results obtained from at least three independent experiments. For imaging analyses, >500 cells were counted in each experiment. (A) Subcellular distributions of CIMPR, TGN46, or TfnR in HeLa cells treated with control nontargeting or mDpy-30 siRNAs. Cells were processed as described in Fig. 1. Arrows indicate cell protrusions. (B) Enrichment of CIMPR near cell protrusions (indicated by arrows) was confirmed by phalloidin F-actin costaining, and the fraction of cells displaying enriched CIMPR near protrusions was scored. (C) Western blot analysis of the CIMPR protein level after the knockdown of mDpy-30. The numbers represent the mean value of six independent experiments. (D) The effect of mDpy-30 overexpression on the localization of CIMPR or β-GalT1 (compare asterisks with arrows). Only those cells exhibiting a high level of exogenous mDpy-30 were counted. The experiment was repeated once, and the mean value is shown. (E) The protein level of CIMPR in cells overexpressing mDpy-30. (B and E) Error bars indicate the standard deviation of the values obtained from independent experiments. Bars, 10 µm.