Gel developer

B16/F10 cells were treated with KGO in the presence of α-MSH (10µM). Protein was extracted from the cells and the mouse skin of the UVB-irradiated mice according to the manufacturer’s instructions for the PRO-PREP^® lysis buffer (iNtRON Biotechnology, Republic of Korea). The preceding steps were performed according to our previously reported study [30 (link)]. Briefly, the protein was separated on 10% SDS-PAGE and transferred onto PVDF membranes, followed by blocking with skim milk. Membranes were washed and incubated with a primary antibody at a dilution of 1:1000 overnight at 4 °C on a roller. The next day, the membranes were washed and incubated with a secondary antibody (1:3000 dilution) for 75 min before developing enhanced chemiluminescence in a gel developer (General Electrics, Boston, MA, USA).

RAW 264.7 cells were treated with Rg3-RGE + PT or left untreated in the presence or absence of LPS (0.1 μg/mL). Later steps were according to our previously reported study [30 (link)]. Briefly, protein was extracted from cells and separated via 10% SDS-PAGE. Proteins were transferred to PVDF membranes, blocked, and incubated with the respective primary antibodies (1:1000). The next day, membranes were washed and incubated with secondary antibody (1:3000) for 75 min before developing in a gel developer (General Electrics, Boston, MA, USA).

Proteins were extracted from 3T3-L1 cells and liver tissues of mice and analyzed using western blot analysis. Cells and liver tissues were homogenized with Pro-Prep protein lysis solution (Invitrogen, Daejeon, Republic of Korea), and protein concentrations were analyzed using the Bradford method. Proceeding procedures were conducted as previously reported [28 (link)]. Briefly, proteins were separated using 10% SDS-PAGE and transferred to a PVDF membrane, followed by blocking with 5% skim milk at room temperature for 1 h. The membranes were then incubated with the respective primary antibodies (1 : 3,000) overnight, followed by incubation with a secondary antibody (1 : 1,000) at room temperature for 90 min. The membranes were then washed with 1% Tween-20 TBS before developing the membranes using ECL chemiluminescence in a gel developer (General Electric, Boston, MA, USA). Western blot analysis was repeated in triplicate, and the relative expressions were quantified using ImageJ software (NIH, USA).