Figure 5
Figure 5. Western blot analysis of matriptase-2 protein in rat liver tissues. (A) Western blot analysis of matriptase-2 (M2), TfR1, and β-actin in rat muscle and whole liver tissues. Gastrocnemius muscle and liver extract proteins (250 μg) from rats fed either a control diet (control) or an ID diet for 3 days were separated by use of 11% SDS-PAGE under reducing conditions, followed by transfer onto nitrocellulose membrane. Membranes were probed with rabbit anti–matriptase-2 (M2), mouse anti-TfR1 (TfR1), and mouse anti–β-actin, followed by immunodetection with corresponding HRP-conjugated secondary antibodies. The blot was exposed to X-ray film. Cell lysates (100 μg protein per lane) from HEK293 cells transiently transfected with either empty vector (C), human TMPRSS6 cDNA (hM2), or rat TMPRSS6 cDNA (rM2) were included as the negative and positive controls, respectively. Two images for matriptase-2 (M2) with different exposure time (top and middle panels) were presented to show the M2 bands in transfected cells. Note that muscle lysates have high levels of β-actin. (B) Western blot analysis of matriptase-2 (M2) and β-actin in mouse liver tissues. Liver membrane extract proteins (250 μg) from 2 8-week-old Tmprss6−/− mice (−/−) and 2 age- and sex-matched wild-type counterparts (wt) were separated with 11% SDS-PAGE under reducing conditions, followed by transfer onto nitrocellulose membrane. Membranes were probed with rabbit anti–matriptase-2 (M2) and mouse anti–β-actin, followed by immunodetection with corresponding HRP-conjugated secondary antibodies. The blot was exposed to X-ray film. Cell lysates (100 μg protein per lane) from HEK293 cells transiently transfected with either empty vector (C) or rat TMPRSS6 cDNA (rM2) were included as the negative and positive controls, respectively. Experiments were repeated twice with consistent results. (C) Western blot analysis of matriptase-2 (M2) and β-actin in HepG2 and HEK293 cells. HepG2 and HEK293 cells in 12-well pates were transiently transfected with either pcDNA3.1-human TMPRSS6 (pcDNA3.1-M2) or pcDNA3.1 empty vector with Lipofectamine 2000. At approximately 48 hours after transfection, cell lysates were subjected to 11% SDS-PAGE, followed by immunodetection with rabbit anti–matriptase-2 (M2), mouse anti–β-actin, and the corresponding HRP-conjugated secondary antibodies. The blot was exposed to X-ray film. Experiments were repeated 4 times with consistent results. (D-F) Western blot analysis of matriptase-2 (M2) and β-actin (actin) in rat liver tissue extract proteins (250 μg) from the animals at 1 day (D), 2 days (E), and 3 days (F). Cell lysates (100 μg protein per lane) from HEK293 cells transiently transfected with either empty vector (C) or human TMPRSS6 cDNA (hM2) were included as the negative and positive controls, respectively. Experiments were performed as described previously in panel A. For each set of analyses, both matriptase-2 (M2) and β-actin (actin) images were derived from the same gel. (G) Quantitative immunoblot analysis of matriptase-2 in panels D through F. The quantification of matriptase-2 bands in panels D through F was performed in essentially the same manner as described previously except that fluorescently labeled goat secondary antibody was used. The intensity of each band was recorded as arbitrary units. We first normalized the intensity of the matriptase-2 band by using β-actin as a loading control. The relative amounts of matriptase-2 in the ID group versus in the corresponding control group are presented. The differences between the control and ID groups were evaluated with the 2-tailed Student t test. All the Western blot analyses were repeated at least twice and showed consistent results. hM2, human matriptase-2; rM2, rat matriptase-2; **P < .01; ***P < .001.

Western blot analysis of matriptase-2 protein in rat liver tissues. (A) Western blot analysis of matriptase-2 (M2), TfR1, and β-actin in rat muscle and whole liver tissues. Gastrocnemius muscle and liver extract proteins (250 μg) from rats fed either a control diet (control) or an ID diet for 3 days were separated by use of 11% SDS-PAGE under reducing conditions, followed by transfer onto nitrocellulose membrane. Membranes were probed with rabbit anti–matriptase-2 (M2), mouse anti-TfR1 (TfR1), and mouse anti–β-actin, followed by immunodetection with corresponding HRP-conjugated secondary antibodies. The blot was exposed to X-ray film. Cell lysates (100 μg protein per lane) from HEK293 cells transiently transfected with either empty vector (C), human TMPRSS6 cDNA (hM2), or rat TMPRSS6 cDNA (rM2) were included as the negative and positive controls, respectively. Two images for matriptase-2 (M2) with different exposure time (top and middle panels) were presented to show the M2 bands in transfected cells. Note that muscle lysates have high levels of β-actin. (B) Western blot analysis of matriptase-2 (M2) and β-actin in mouse liver tissues. Liver membrane extract proteins (250 μg) from 2 8-week-old Tmprss6−/− mice (−/−) and 2 age- and sex-matched wild-type counterparts (wt) were separated with 11% SDS-PAGE under reducing conditions, followed by transfer onto nitrocellulose membrane. Membranes were probed with rabbit anti–matriptase-2 (M2) and mouse anti–β-actin, followed by immunodetection with corresponding HRP-conjugated secondary antibodies. The blot was exposed to X-ray film. Cell lysates (100 μg protein per lane) from HEK293 cells transiently transfected with either empty vector (C) or rat TMPRSS6 cDNA (rM2) were included as the negative and positive controls, respectively. Experiments were repeated twice with consistent results. (C) Western blot analysis of matriptase-2 (M2) and β-actin in HepG2 and HEK293 cells. HepG2 and HEK293 cells in 12-well pates were transiently transfected with either pcDNA3.1-human TMPRSS6 (pcDNA3.1-M2) or pcDNA3.1 empty vector with Lipofectamine 2000. At approximately 48 hours after transfection, cell lysates were subjected to 11% SDS-PAGE, followed by immunodetection with rabbit anti–matriptase-2 (M2), mouse anti–β-actin, and the corresponding HRP-conjugated secondary antibodies. The blot was exposed to X-ray film. Experiments were repeated 4 times with consistent results. (D-F) Western blot analysis of matriptase-2 (M2) and β-actin (actin) in rat liver tissue extract proteins (250 μg) from the animals at 1 day (D), 2 days (E), and 3 days (F). Cell lysates (100 μg protein per lane) from HEK293 cells transiently transfected with either empty vector (C) or human TMPRSS6 cDNA (hM2) were included as the negative and positive controls, respectively. Experiments were performed as described previously in panel A. For each set of analyses, both matriptase-2 (M2) and β-actin (actin) images were derived from the same gel. (G) Quantitative immunoblot analysis of matriptase-2 in panels D through F. The quantification of matriptase-2 bands in panels D through F was performed in essentially the same manner as described previously except that fluorescently labeled goat secondary antibody was used. The intensity of each band was recorded as arbitrary units. We first normalized the intensity of the matriptase-2 band by using β-actin as a loading control. The relative amounts of matriptase-2 in the ID group versus in the corresponding control group are presented. The differences between the control and ID groups were evaluated with the 2-tailed Student t test. All the Western blot analyses were repeated at least twice and showed consistent results. hM2, human matriptase-2; rM2, rat matriptase-2; **P < .01; ***P < .001.

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