Phosphor smad2 3

NHDFs were treated with UVB irradiation (144 mJ/cm²) and PVE (1, 10, 100 μg/mL) and then checked for alterations in the level of signaling molecules in the MAPK/AP-1, NF-κB, and TGF/Smad pathways, respectively. Cells were lysed using RIPA Lysis buffer (Cell Signaling Technology, Danvers) and the protein concentration was measured using Bradford reagent (Bio-Rad, Hercules, CA) as described by the manufacturer. The proteins were separated on 10% SDS-PAGE and transferred onto PVDF membranes. The membrane was incubated with various primary antibodies after blocking. Protein bands were visualized with enhanced chemiluminescence detection reagents after hybridization with HRP-conjugated secondary antibodies. Antibodies against ERK, phosphor-ERK, JNK, phosphor-JNK, p38, and phosphor-p38, as well as anti-rabbit-HRP and anti-mouse-HRP antibodies were purchased from Cell Signaling Technology, and those against NF-κB p65, c-Fos, phosphor-c-Fos, c-Jun, phosphor-c-Jun, TGF-β1, Smad2/3, phosphor-Smad2/3, Smad7, and β-actin were purchased from Santa Cruz Biotechnology (Dallas). Each experiment was repeated at least three times.