Hrp conjugated secondary antibodies

LPS was purchased from Sigma (MO, USA). Anti‐Sirt3, anti‐Cyt‐c and anti‐acetylated lysine were obtained from Cell Signalling Technology (MA, USA). Immunofluorescent Anti‐Sirt3 was purchased from Santa Cruz (CA, USA). Anti‐OPA1, anti‐Drp1, anti‐VDAC, anti‐TOM‐20 and anti‐GAPDH were purchased from Abcam (MA, USA). Anti‐Bax and anti‐YME1L1 were purchased from Proteintech (Wuhan, China). Anti‐Flag, Alexa Fluor 488‐ and 594‐conjugated fluorescent secondary antibodies and HRP‐conjugated secondary antibodies were purchased from Antgene (Wuhan, China).

Flocculus tissue was isolated on ice and homogenized in an ice‐cold RIPA Lysis Buffer (Beyotime) with proteinase inhibitors. The supernatant was collected by centrifugation at 12,000 rpm for 15 min at 4°C. Protein concentration was determined by using an Enhanced BCA Protein Assay Kit (Beyotime). Protein lysates (20 μg) were separated on 12% SDS‐polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked and incubated with primary antibodies overnight at 4°C. The primary antibodies used were mouse‐anti‐mGluR1α (1:1000, BD Pharmingen, Code No: 556389), rabbit‐ERK (1:1000, Immunoway, Code No: YT1625), rabbit‐p‐ERK (1:1000, Immunoway, Code No: YP0101), rabbit‐α‐Tubulin (1:4000, Abclonal, Code No: AC003), and rabbit‐anti‐β‐actin (1:5000, Antgene, Code No: ANT321). The membranes were then incubated with HRP‐conjugated secondary antibodies (1:3000, AntGene) and washed to remove excess primary antibody. Bands were detected by employing BeyoECL Plus (Beyotime) and digitally quantified by utilizing Image‐Pro Plus 6.0 software (Media Cybernetics, Inc.).