Bs 23687r

After treatment, the cells were washed with cold PBS and lysed in lysis buffer (Biotopped, Beijing, China) with the protease inhibitor (Biotopped). The cell lysates were placed on ice for 30 min, and then centrifuged at 12,000 rpm for 10 min at 4 °C to collect the supernatant. The protein concentrations in the supernatant were quantified using a BCA protein assay kit (Biomed, Beijing, China), and sample proteins were separated by SDS-PAGE and transferred onto an NC membrane (Biotopped). The membranes were blocked with 5% nonfat dry milk in Tris aminoethane tween (TBST) buffer (Biotopped) for 2 h and then incubated with primary antibodies: SIRT2 (1:500, #19655-1-AP; Proteintech), Tomm20 (1:500, #11802-1-AP; Proteintech), Parkin (1:500, #bs-23687R; Bioss), p-ERK1/2 (Thr202/Tyr204, 1:1000, #9101; CST, Danvers, MA, USA), ERK1/2 (1:1000, #9101; CST), GAPDH (1:10000, #A01020; Abbkine, CA, USA), and β-actin (1:1000, #ab8226; abcam, Cambridge, England, UK) at 4 °C overnight. Following washing with TBST, membranes were incubated with related horseradish-peroxidase-conjugated secondary antibodies at room temperature for 1 h. Membranes were then incubated in ECL reagents (Biotopped) and images were captured by a chemiluminescence imaging analyzer.

Cell slides were added to 6-well plates before cell seeding, and then, a total of 1 × 10⁵ cells were seeded on the slides with cell culture medium. When the cells reached a confluence of 70–80%, the 6-well plates were washed with PBS 3 times. Then, the cells were fixed with 4% paraformaldehyde for 20 min, washed 3 times with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 30 min. Then, blocking with 3% BSA was performed for 1 h at room temperature, and polyclonal rabbit anti-PINK1 (1:200, #23274-1-AP; Proteintech, Wuhan, China), rabbit anti-Parkin (1:200, # bs-23687R; Bioss, Beijing, China), and rabbit anti-LC3 (1:200, #18725-1-AP; Proteintech) antibodies were applied, and the cells were incubated overnight at 4 °C. After washing, cells were incubated with an Alexa Fluor 488-conjugated secondary antibody (1:300, #A-11034; ThermoFisher Scientific, Waltham, MA, USA) for 1 h. Then, the plates were washed three time with PBS, and stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China) for 6 min. Finally, images were obtained using a fluorescence microscope (Olympus).

Brain tissue (5 μm) were cut and fixed in formalin buffer after being paraffin-embedded. Paraffin-embedded sections of brain tissue were first deparaffinized and dehydrated. Slides were incubated with 0.3% H₂O₂ for 10 min and washed with ddH₂O three times, 3 min each time. Antigen retrieval was undertaken in a pressure cooker for 10 min with 0.01 M citrate buffer and washed with phosphate-buffered saline (PBS) three times, 5 min each time. Five percent bovine serum albumin (BSA) was applied to the specimen sections to block nonspecific protein for 20 min at room temperature. The primary antibodies, which were rabbit antibodies against Beclin1 (bs-1353R, Bioss, China), Parkin (bs-23687R, Bioss, China), PINK1 (6946, CST, USA), or BNIP3L/NIX (12396, CST, USA), were incubated for 2 h at 37°C and washed with PBS three times, each time 3 min. After washing, slides were washed and then incubated with secondary antibody, goat anti-rabbit IgG (ab7090, Abcam, UK), at 37°C for 30 min and washed with PBS three times, 3 min each time. The sections were stained with diaminobenzidine for ~5–10 min. Five nonoverlapping visual fields at 400× magnification were analyzed from each sample using a Moticam 3000 (Hong Kong Special Administrative Region, China) microphotography system. The mean number of positive cells was calculated using Image-Pro Plus 6.0 software (Xue et al., 2014 (link)).