Figure 6
Figure 6. Influence of PRCP depletion in cultured cells. (A) HUVECs were treated with control (CN) or PRCP siRNA overnight, and comparative PRCP expression levels were analyzed by immunoblot. Data are mean ± SEM of 3 samples. (B) DHE fluorescent images of siRNA-treated HUVECs were obtained after control (CN; n = 6) or PRCP knockdowns (n = 6). Data are a representative experiment on siRNA-treated cells, and the adjacent graph is the mean ± SEM of pixel density per nucleus measured by morphometric analysis of 6 experiments for each group. (C) DHE fluorescent image of MEFs from control (n = 10) and PRCPgt/gt (n = 11) embryos. The figure presented is a representative experiment on each of the cells, and the adjacent graph is the mean ± SEM of pixel density per nucleus measured by morphometric analysis of ≥ 10 experiments for each condition. (B-C) Photographed at 40× on a Nikon, TE200 immunofluorescent microscope aperture 0.75/0.17 WD 0.72 oil immersion. The relative DHE fluorescence was measured by morphometric analysis on 40× microscopic images using MetaMorph (Molecular Devices). (D) Protein C activation on HUVECs with control (CN) or PRCP siRNA treatment as measured by activated protein C hydrolysis of a chromogenic substrate. Data presented are the percentage activated protein C hydrolysis generated on HUVECs treated with PRCP siRNA versus control. Data are mean ± SEM of 6 experiments per condition. (E) Thrombomodulin (TM) in HUVECs treated with control (CN) or PRCP siRNA as seen on immunoblot. Data presented are the mean ± SEM ratio of TM antigen/GAPDH antigen of 4 experiments. (F) mRNA expression for eNOS from control (CN) or PRCP knockdown siRNA-treated HUVECs as measured by real-time PCR (control: n = 7, PRCP knockdown: n = 7). (G) An immunoblot for eNOS was performed on control and PRCP siRNA-treated HUVEC lysates. The normalized relative ratio of uncoupled:coupled eNOS in lysates from 3 samples of control or PRCP siRNA-treated cells were compared by analysis of band intensity using densitometer scanning. Immunoblot for GAPDH was used as a loading control. *P < .05.

Influence of PRCP depletion in cultured cells. (A) HUVECs were treated with control (CN) or PRCP siRNA overnight, and comparative PRCP expression levels were analyzed by immunoblot. Data are mean ± SEM of 3 samples. (B) DHE fluorescent images of siRNA-treated HUVECs were obtained after control (CN; n = 6) or PRCP knockdowns (n = 6). Data are a representative experiment on siRNA-treated cells, and the adjacent graph is the mean ± SEM of pixel density per nucleus measured by morphometric analysis of 6 experiments for each group. (C) DHE fluorescent image of MEFs from control (n = 10) and PRCPgt/gt (n = 11) embryos. The figure presented is a representative experiment on each of the cells, and the adjacent graph is the mean ± SEM of pixel density per nucleus measured by morphometric analysis of ≥ 10 experiments for each condition. (B-C) Photographed at 40× on a Nikon, TE200 immunofluorescent microscope aperture 0.75/0.17 WD 0.72 oil immersion. The relative DHE fluorescence was measured by morphometric analysis on 40× microscopic images using MetaMorph (Molecular Devices). (D) Protein C activation on HUVECs with control (CN) or PRCP siRNA treatment as measured by activated protein C hydrolysis of a chromogenic substrate. Data presented are the percentage activated protein C hydrolysis generated on HUVECs treated with PRCP siRNA versus control. Data are mean ± SEM of 6 experiments per condition. (E) Thrombomodulin (TM) in HUVECs treated with control (CN) or PRCP siRNA as seen on immunoblot. Data presented are the mean ± SEM ratio of TM antigen/GAPDH antigen of 4 experiments. (F) mRNA expression for eNOS from control (CN) or PRCP knockdown siRNA-treated HUVECs as measured by real-time PCR (control: n = 7, PRCP knockdown: n = 7). (G) An immunoblot for eNOS was performed on control and PRCP siRNA-treated HUVEC lysates. The normalized relative ratio of uncoupled:coupled eNOS in lysates from 3 samples of control or PRCP siRNA-treated cells were compared by analysis of band intensity using densitometer scanning. Immunoblot for GAPDH was used as a loading control. *P < .05.

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