Enhanced chemiluminescence detection kit

Total protein lysates from the cultured cells were extracted using a lysis buffer containing proteinase inhibitors (Sigma-Aldrich, Darmstadt, Germany). Proteins were denatured, separated in 10% polyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes for probing with the following primary antibodies: anti-NFIB (1:500, Abcam, ab186738, Cambridge, UK), PINK1 (1:500, Abcam, ab216144, Cambridge, UK) and anti-GAPDH (1:2000, Proteintech, 10494-1-AP, Wuhan, China). For detection, anti-rabbit secondary antibodies conjugated to horseradish peroxidase (1:3000, Proteintech, Wuhan, China) were used. Band signals were visualized by an enhanced chemiluminescence detection kit (Meilunbio, Dalian, China). All western blotting was performed in triplicate.

Total protein was extracted from imDCs, mDCs and Met-mDCs using RIPA buffer containing protease (phenylmethylsulfonylfluoride (PMSF)) and phosphatase inhibitors (Solarbio, Beijing, China). The protein concentration was determined by a BCA kit (Servicebio, Beijing, China). Equal amounts of denatured protein samples were separated on a 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gel and transferred onto 0.45 μm PVDF membranes (Merck Millipore Ltd., Tullagreen, Carrigtwohill Co. Cork, Ireland). After being blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against GAPDH, pFoxO3a (s253) and Foxo3a overnight at 4 °C and then with HRP-conjugated secondary antibodies for 1 h at room temperature. The signals were visualized using an enhanced chemiluminescence detection kit (Meilunbio, Dalian, China) in an imaging system (Bio-Rad, Hercules, CA, USA). The mean gray value of each band was analyzed by ImageJ software.

Total protein was extracted from imDCs, mDCs and Met-mDCs using RIPA buffer containing protease (phenylmethylsulfonylfluoride (PMSF)) and phosphatase inhibitors (Solarbio, Beijing, China). The protein concentration was determined by a BCA kit (Servicebio, Beijing, China). Equal amounts of denatured protein samples were separated on a 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gel and transferred onto 0.45 μm PVDF membranes (Merck Millipore Ltd., Tullagreen, Carrigtwohill Co. Cork, Ireland). After being blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against GAPDH, pFoxO3a (s253) and Foxo3a overnight at 4 °C and then with HRP-conjugated secondary antibodies for 1 h at room temperature. The signals were visualized using an enhanced chemiluminescence detection kit (Meilunbio, Dalian, China) in an imaging system (Bio-Rad, Hercules, CA, USA). The mean gray value of each band was analyzed by ImageJ software.