2.5. Western Blotting

Protein levels were investigated using immunoblotting according to common Western blotting protocols. HaCaT cells cultured in 6-well plates were treated with or without DCEQA (1, 5, 10 μM) for 24 h after UVB (15 mJ/cm²) irradiation. Following incubation, wells were aspirated and cells were lysed by vigorous pipetting in 1 ml of RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) at 4°C. The protein content of the lysates was measured with a BCA protein assay kit (Thermo Fisher Scientific) following the kit's protocol. The same amount (20 μg) of protein from each well was loaded onto 12% SDS-polyacrylamide gel and run at 100 V. Proteins on the gel were then transferred onto a polyvinylidene fluoride membrane (Amersham; GE Healthcare, Little Chalfont, UK) using a wet system run at 100 V for 1 h at 4°C. Membranes were then incubated for 1 h at room temperature in 5% skimmed milk for blocking. Blocked membranes were washed with 1X TBST and incubated with primary antibodies against MMP-1 (sc-6837; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP-2 (#4022; Cell Signaling Technology, Beverly, MA, USA), MMP-9 (#393857; Cell Signaling Technology), type I procollagen (sc-8782; Santa Cruz Biotechnology), p38 (#8690; Cell Signaling Technology), phospho (p)-p38 (#4511; Cell Signaling Technology), JNK (LF-PA0047; Thermo Fisher Scientific), p-JNK (sc-293136; Santa Cruz Biotechnology), ERK (#4695; Cell Signaling Technology), p-ERK (#4370; Cell Signaling Technology), and β-actin (sc-47778; Santa Cruz Biotechnology) in primary antibody dilution buffer containing 1X TBST with 5% bovine serum albumin, overnight at 4°C. Membranes were then incubated with horseradish-peroxidase-conjugated secondary antibodies (diluted 1 : 1,000) specific to the primary antibody source organism at room temperature for 1 h. Detection of proteins on blotted membranes was achieved using an ECL Western blot detection kit (Amersham) according to the manufacturer's instructions. Protein bands were imaged with CAS-400SM Davinch-Chemi imager™ (Davinch-K) and bands were quantified from images densitometrically.