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. 2021 Feb 14;22(4):1895. doi: 10.3390/ijms22041895

Figure 6.

Figure 6

Evaluation of receptors and signals involved in melanogenesis. (A) B16F10 cells were exposed to LTAP for 1, 3, or 5 min, and incubated further for 24 h. The protein levels of endothelin 1 (EDN1), melanocortin 1 receptor (MC1R), and Wingless-type protein (WNT) were detected by Western blotting. β-actin was used as a loading control. (B,C) Cells were exposed to LTAP for 5 min and incubated for 3 h. Then 100 nM alpha-melanocyte stimulating hormone (α-MSH) was added and incubated for further 24 h and 15 min. Protein expression levels of MC1R were determined after 24 h of α-MSH treatment. Phosphorylation of protein kinase A (PKA), cAMP response element-binding protein (CREB) was evaluated after 15 min of α-MSH treatment. Phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun-N-terminal kinase (JNK) was evaluated by Western blotting after 30 min of α-MSH treatment. (D) B16F10 cells and NHEMs were harvested after 72 h and the expression of Cyclin A, B1, D1, E1, and Cyclin-dependent kinase (CDK) 1 and CDK2 was evaluated by Western blotting. Numerical values on the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments.