Western blot analysis

To determine the level of type I collagen secreted from control and CHX-treated MEFs, western blot analysis was performed on the cell lysate and conditioned media according to Shi et al. [17]. The samples were mixed with 4×SDS reducing sample buffer containing 40% glycerol, 250 mM Tris-HCl, pH 6.8, 8% sodium dodecyl sulfate, 0.04% bromophenol blue, and 20% 2-mercaptoethanol. The samples were then heated at 100°C for 10 min and subjected to 7.5% or 10% SDS-PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane (HATF00010; Merck Millipore, Billerica, USA), which was blocked with 5% BSA in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) at room temperature (RT) for 3 h and then incubated with anti-type I collagen antibody (0.4 μg/mL), anti-P4HA1 (1 μg/mL), anti-P4HA2 (1 μg/mL), or anti-actin antibody (0.2 μg/mL) overnight at 4°C. Bound primary antibodies were detected with HRP-conjugated rabbit anti-goat, goat anti-rabbit or anti-mouse IgG secondary antibodies (0.12 μg/mL). The amount of type I collagen was quantified by densitometry using ImageJ software (NIH) and was normalized to actin loading controls.