Ripa lysis buffer kit

As described previously,², ⁷ cell lysates were prepared from FLS with RIPA lysis buffer kit (Beyotime Biotechnology), and the protein concentrations were quantified using a BCA protein assay kit (Thermo Scientific). Next, 8%–15% SDS‐PAGE gels were used to separate the cell lysates, and the samples were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Subsequently, the membranes were incubated with primary antibodies anti‐IL‐1β, IL‐6, TNF‐α, TLR4, MyD88 (Abcam) and β‐actin (Proteintech) overnight at 4°C. After washing thrice with TBST, the membranes were incubated with Horseradish Peroxidase (HRP)‐labelled secondary antibodies (Proteintech) for 1 h at room temperature. Images were developed after reaction with a high‐sensitivity chemiluminescence reagent (Proteintech).

Total protein was extracted using the RIPA lysis buffer kit (Beyotime, Shanghai, China), and total protein was mixed with 1x loading buffer. 10% SDS-PAGE gel electrophoresis was performed, and samples were transferred onto PVDF membranes (Millipore Corp. Billerica, MA, USA). Then, membranes were incubated with 5% skim milk and incubated with primary antibodies overnight at 4°C. Membranes were then incubated with secondary peroxidase-conjugated antibodies for 1 h at room temperature. Finally, a SuperSignal chemiluminescent substrate (Millipore, Billerica, MA, USA) was used to visualize protein bands. Primary antibodies for this study were specific for CA12 (Boster, Cat. No. A04063, 1 : 1000), LONRF3 (GeneTex, Cat. No. GTX112150, 1 : 2000), MAP2 (Boster, Cat. No. A01201, 1 : 2000), THBS1 (Boster, Cat. No. PB0471, 1 : 2000), PPID, E-cadherin (Beyotime, Cat. No. AF6759, 1 : 1000), N-cadherin (Beyotime, Cat. No. AF5237, 1 : 800), aggrecan (Abcam, Cat. No. ab3778, 1 : 1000), Col2A1 (Boster, Cat. No. A00517, 1 : 2000), MMP13 (Proteintech, Cat. No. 18165-1-AP, 1 : 3000), ADAMTS4 (Proteintech, Cat. No. 11865-1-AP, 1 : 600), and GAPDH (Abcam, Cat. No. ab9485, 1 : 2000). GAPDH was used as internal control.

According to the user-instruction of the RIPA Lysis Buffer Kit (Beyotime, Shanghai, China), 100 µL of lysis buffer was added to 1 × 10⁶ peritoneal cells pellet or 10 mg tissue. After ultrasonication for 20 min, the lysate was centrifuged at 12000 × g for 30 min, and then the protein concentration in the supernatant was adjusted to 1mg mL^-1 by using the BCA kit (EpiZyme, Shanghai, China). Twenty microliters of the cell or tissue (head kidney, spleen, gill, skin, muscle, and liver) lysates went through SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). Then the membranes were blocked with 5% BSA and incubated with anti-rPoMPO, anti-Trx, or anti-GAPDH Abs (latter ab: Abclonal, Wuhan, China). After washing three times with PBST, the membranes were incubated with goat anti-mouse IgG-HRP or goat anti-rabbit IgG-HRP (Sigma, St. Louis, Mo, USA) diluted with 5% BSA at 37 °C for 45 min. Finally, the bands were detected with ECL Enhanced Kit (Abclonal, Wuhan, China) in a chemiluminescence detection instrument (Vilber, Ile-de-France, France). The semi-quantification of protein bands from homogenized PerCs priority subjected to LPS (100 µg per individual (N = 3)), Sigma, St. Louis, Mo, USA) at different time points was analyzed using Image J software (34 (link)).