Bcl2 associated x (bax)

Skin tissues and cells were lysed with the RIPA lysis buffer (Servicebio, Wuhan, China) containing protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). The protein concentration was measured by BCA assay (Beyotime Biotechnology, Shanghai, China). Lysates (20–40 μg total protein) were resolved by SDS-PAGE. The extracts were then transferred onto a PVDF membrane (Merck Millipore, Billerica, MA). The following primary antibodies were used: pPDE4D (1:500; Abcam, Cambridge, UK); pCREB (1:500), ERK (1:1,000) and phospho-ERK (pERK, 1:500) from Cell Signaling Technology (Danvers, MA, United States); CREB (1:1,000) and Bax (1:1,000) from Bimake (Houston, TX, United States); K17 (1:500; Santa Cruz Biotechnology, United States); PDE4D (1:500), Bcl2 (1:200), and GAPDH (1:10,000) from Proteintech (Chicago, IL, United States). Chemiluminescent detection was performed with horseradish peroxidase–coupled secondary antibody from Proteintech (Chicago, IL, United States) and an ECL chemiluminescence reagent kit (Beijing Labgic Technology, China). Band densities were quantified using ImageJ software.

After the treatment of cells with 6-OHDA and vitamin K2, respectively, cells were lysed with a lysis buffer (Beyotime) and centrifuged at 4 °C for 10 min at 12,000 rpm. A BCA assay kit (Sangon Biotech, Shanghai, China) was used to measure the concentration of total protein. For each group, 20 μg protein total proteins were separated by using 12% SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocked with protein-free rapid blocking buffer (Epizyme Biotech, Shanghai, China) at 22 ± 3 °C for 20 min, the membranes were incubated with primary antibodies, including PGC-1α, PINK1, MFN1, MFN2, Nrf2, Bax, TFAM (Bimake, Houston, TX, USA), Bcl-2 (Cell Singling Technology, Boston, MA, USA) and β-actin (Proteintech, Wuhan, China), respectively, at appropriate dilutions, overnight, at 4 °C. After washing with TBST for 30 min, the membranes were incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (Proteintech) at 22 ± 3 °C for 1 h. Finally, the PVDF membranes were then covered with a hypersensitive enhanced chemiluminescence (ECL) solution (Vazyme) and visualized by using a luminescent image analyzer (UVITEC, Mini HD9, Cambridge, UK).