West pico chemiluminescent kit

Total cellular protein extracts were prepared on ice using a PRO-PREP protein extract solution (Intron, Seoul, Korea). Cell lysates were loaded onto a sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene fluoride membrane for 1 hour. Membranes were incubated at 4 °C overnight with HIF-1α, apo-A4, TNFR2, IκB, and α-tubulin primary antibodies (Abcam, Cambridge, MA, USA) diluted 1:1000 with 5% BSA Tris-buffered saline-Tween 20 (TBS-T). After incubation, the membranes were washed with TBS-T and then secondary antibodies (1:10000, horseradish peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG) were added for incubation at room temperature for 1 hour. Labeled bands were detected by West Pico chemiluminescent kit (Thermo Scientific, Rockford, IL, USA).

HaCaT cells were cultured in 6-well plates, treated with Perilla leaf or callus extracts for 12 h, UVB-irradiated, and then incubated for 3 h at 37 °C [14 (link)]. Following incubation, cells were harvested with trypsin-EDTA, resuspended in lysis buffer on ice for 40 min, centrifuged at 13,714× g for 20 min at 4 °C, and protein concentrations in supernatants were determined using a NanoDrop (NanoDrop Lite spectrophotometer, Waltham, MA, USA). Equal amounts of proteins were subjected to SDS-PAGE and then transferred to nitrocellulose membranes, which were reacted with primary antibodies; pCHK1 (Serine 345), γH2AX (both from Cell Signaling, Danvers, MA, USA), CyclinD1, Cell division protein kinase 6 (CDK6), and β-actin (all antibodies from Santa Cruz Bicycles, Santa Cruz, CA, USA) at 4 °C overnight. The following day, membranes were incubated with anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies for 40 min at room temperature. Blots were detected using the West Pico chemiluminescent kit (Thermo, Rockford, IL, USA) and visualized using LAS 4000 chemiluminescent image analyzer (Fuji, Tokyo, Japan).