Ar0102

Chondrocytes were inoculated into six-well plates at a density of 5 × 10⁵ cells per well and allowed to adhere for 48 hours. First, the cells were treated in different groups for 24 hours. The cells were washed twice with phosphate-buffered saline (PBS), and then RIPA lysis buffer containing a 1% protease inhibitor mixture (AR0102; Boster, China) was used to lyse chondrocytes for 30 minutes on ice. The extract was collected and subjected to centrifugation at a speed of 12,000× g and a temperature of 4°C for 30 minutes. Then, a BCA analysis kit (AR0146; Boster) was used to determine the protein concentration of cell lysates. Next, the supernatant of each sample was collected, and then the samples (25 μg) were separated through electrophoresis on a 12% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (MilliporeSigma, USA). After being blocked with 5% skimmed milk at ordinary temperature for one hour, the membranes and specific primary antibodies were incubated at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies at room temperature for one hour. To visualize the protein bands, ultra-sensitive ECL chemiluminescence reagent from Boster was employed. The resulting images were captured using the ChemiDocTM XRS+ System (Bio-Rad Laboratories, USA).

Immediately after the rats were euthanized (Figure 1B), their mPFC, CeA, and hippocampal CA1 were retrieved immediately through dissection and stored at –80°C until analysis (Li et al., 2016 (link), 2017 (link)). The tissues were homogenized and lysed using a radio-immuno-precipitation assay buffer (AR0102, Boster) and then subjected to the measurement of protein concentration using the bicinchoninic acid kit (AR1189, Boster). The lysates (20 μg each) were separated on 10% SDS-PAGE gels, and the separated protein bands were transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane. Immunoreactivity was determined based on enhanced chemiluminescence, and the signals were detected using a Bio-Rad ChemiDoc system (Bio-Rad Laboratories, China).
The following antibodies were used: anti-GAPDH (1:5,000, BM1623, Boster); anti-GluA1 (1:1,000, A1826, ABclonal); anti-mGluR1 (1:1,000, abx112750, Abbexa); anti-mGluR5 (1:1,000, A3758, ABclonal); anti-GluN2B (1:1,000, abx23583, Abbexa); anti-PSD-95 (1:1,000, abx236850, Abbexa); anti-α5 GABA (1:1,000, ab259880, Abcam). The secondary antibodies used were goat anti-rabbit IgG (1:10,000; Boster) and goat anti-mouse IgG (1: 10,000, Boster). The immunosignals were quantified using densitometry and were expressed relative to the GAPDH signals and normalized to the control for data analysis.

Lung tissue was added to an RIPA lysate (AR0102, Boster, Wuhan, China) premixed with PMSF and a phosphatase inhibitor (ST506, Beyotime, Shanghai, China) and homogenized thoroughly. It was then placed on ice for 30 min and centrifuged at 4 °C and 12,000 rpm for 10 min to obtain the proteins. These proteins were assayed for concentration using a BCA kit (P0010S, Beyotime, Shanghai, China) and mixed with a loading buffer (WB2001, NCM Biotech, Suzhou, China) to make protein samples. After SDS-PAGE gel electrophoresis, the proteins were separated according to their molecular weight and size. After the membrane transfer operation, the protein samples were enriched on the PVDF membrane. Subsequently, the PVDF membrane containing the target proteins was blocked with a 5% BSA solution and sequentially incubated with primary and secondary antibodies. Finally, images were captured on a gel imaging system in conjunction with ECL (P10060, NCM Biotech, Suzhou, China) reagents. The antibodies are provided in Table 2.