Figure 1

Analysis of extracellular collagen and intracellular HSP47 proteins in ZF4 cells cultured at different temperatures. (a) The loaded amount of SDS samples prepared from culture media was normalized to the protein amount of the cell layer. SDS samples are loaded onto SDS-PAGE gel (5%) under reducing conditions. After transfer to a nitrocellulose membrane, collagen polypeptides were detected by using biotinylated soCMP6-7(Glu)2. (b) The culture media of ZF4 cells was treated with pepsin at 4 °C, and the triple-helical region of collagen was purified through salt precipitation. SDS-PAGE (5%) was performed under nonreducing conditions. Proteins were visualized with CBB. (c) HSP47 was pulled down from the lysate of ZF4 cells using type I collagen-coupled beads or mock-coupled beads. The SDS samples were subjected to SDS-PAGE (10%) under nonreducing conditions, and western blotting was performed using an antibody against HSP47 (top). The same membranes were reprobed with the anti-β-actin antibody (bottom).