Figure 6.

AZGP1 inhibits TGF-β–induced Smad and ERK phosphorylation, which is linked to its internalization by endocytosis. (A) Smad reporter luciferase assay in NRK-49F cells after 24 hours of TGF-β stimulation (2 ng/ml) in the presence or absence of recombinant AZGP1 (25 ng/ml). (B and C) Representative Western blots for phosphorylated Smad2, Smad3, Smad1/Smad5/Smad9, ERK, and their respective total protein after 24 hours of TGF-β stimulation (2 ng/ml) in the presence or absence of recombinant AZGP1 (25 ng/ml). (D) Representative confocal microscopy image showing NRK-49F cells at 2 hours after administration of FITC–labeled recombinant human AZGP1. Cellular uptake of labeled AZGP1 is significantly reduced by (E and F) pretreatment with genistein (200 µM) and (G and H) after siRNA-mediated knockdown of caveolin-1. (I) Quantification of cytoplasmic FITC–positive pixels after treatment with control or anticaveolin-1 siRNA. Quantitative RT-PCR shows change in AZGP1 effect on αSMA expression (J and K) after pretreatment with genistein (200 µM) and (L and M) after siRNA-mediated knockdown of caveolin-1. Values are given as means±SEMs (n=3 independent experiments for each data point in A, I, and J–M.) Original magnifications, ×63 in D–H. *P<0.05; **P<0.005; ***P<0.001.