Sb203583

(3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) was obtained from AMRESCO (Solon, OH, USA). Compound K (purity: > 96%), L-3,4-dihydroxyphenylalanine (L-DOPA), α-melanocyte-stimulating hormone (α-MSH), arbutin, and mushroom tyrosinase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco (Grand Island, NY, USA). Inhibitors (SB203583, SP600125, and U0126) were purchased from Millipore (Billerica, MA, USA). Total and phospho-specific antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho- and total antibodies against p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), inhibitor of κBα (IκBα), and β-actin were purchased from Cell Signaling (Beverly, MA, USA).

HSFs were cultured in 100 mm Petri dishes. The inhibitor group was first treated with the small-molecule inhibitors SB431542, LY294002, AG490 or PD98059 for 2 h (10 μM). The inhibitors SB203583 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (an ERK1/2 inhibitor), AG490 (a JAK inhibitor) and BX795 (a TBK1 inhibitor) were purchased from Millipore (Billerica, MA). Then, the cells were treated with 3D-GF-PADM at 1 mg ml⁻¹ for 24 h. After collection, total protein extraction was done with RIPA buffer containing phosphatase and protease inhibitors (Pierce, Rockford, IL, USA). After separation using a 12% SDS-PAGE denaturing gel and transfer to PVDF membranes, the membranes were blocked with 5% skim milk. The primary antibodies, including JAK2 (Abcam; ab108596), p-JAK2 (Abcam; ab32101), STAT3 (Abcam; ab68153), p-STAT3 (Abcam; ab76315) and HAS2 (Abcam; ab140671), were used at 4 °C overnight. Secondary antibody incubation was performed at room temperature for 1 h.