Figure 5.
(A-E) Silencing of CD151 protein decreased phosphorylation status and nuclear transport of SMAD3. (A) Immunoblotting demonstrated little or no pSMAD3 in CD151 knockout HS-MMhigh cells. The intensity of the GAPDH reference did not change. Semi quantitation was performed as described in the Materials and methods. The intensity ratio of each band to GAPDH is shown. The data represent mean ± SD in triplicate assays. Student's t-test was performed to confirm the significant differences, **P<0.01 vs. counterpart. The pSMAD3/SMAD3 ratio was 0.17±0.07 (Mock) and 0.016±0.007 (CD151 downregualtion group) in triplicate assay (Student's t-test, P<0.01). (B and D) HS-MMhigh cells exhibited CD151 immunoreactivity at the cell membrane surface (green staining; Alexa Fluor 488) and pSMAD3 was detected in both the cytoplasm and nucleus (red staining; Alexa Fluor 555). (C and E) Downregulation of CD151 diminished immunoreactivity of CD151 or pSMAD immunoreactivity. All nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 10 µm. Original magnification, ×100 (B and C) and ×400 (D and E). The results shown were obtained using SI02777257 siRNA (Qiagen) to silence the CD151 gene. Similar results were obtained using SI02777250 siRNA. pSMAD3, phospho-SMAD family member 3; CD151, cluster of differentiation 151; SD, standard deviation.