Beyoecl moon

According to the GenBank database, the siRNA sequence(TableS2) of rat and human ENO1 gene designed and obtained from JTSBIO Co,Ltd (Wuhan, China). ENO1-siRNA and negative control (NC) vectors were transfected into primary cultured FLS cells of rat and human.
The knees of rat models were embedded in paraffin, sectioned at a thickness of 5-μm, and then stained with hematoxylin and eosin. Cells were lysed using RIPA buffer (Beyotime, China). Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight. Membrane was subsequently incubated with HRP-conjugated secondary antibodies. BeyoECL Moon (Beyotime, China) was used to visualize the protein bands. Detection was performed using an automated chemiluminescence image analysis system (SyngeneG Box chemi xx6, British) and the quantification of the bands was performed using Image J software.

H9C2 cells with different treatment were lysed, quantified and subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes (Cat # 1,620,177, Bio-Rad, Hercules, CA, USA) which were incubated with 5% nonfat milk in TBST and followed by incubation of primary antibodies at 4°C overnight. Then the PVDF membranes were washed with TBST and incubated with secondary antibodies. Protein expression was detected using BeyoECL Moon (Cat # P0018FS, Beyotime) under a Tanon 5200 machine (Shanghai, China). The detailed primary and secondary antibodies were listed as below: anti-cleaved caspase 3 (Cat # E83-77, Abcam); anti-caspase 3 (Cat # ab32351, Abcam); anti-Bax (Cat # WL01637, Wanleibio, Shenyang, China); anti-Bcl-2 (Cat # WL01556, Wanleibio); anti-β-actin (Cat # WL01372, Wanleibio); anti-Nrf2 (Cat # WL02135, Wanleibio); anti-Sirt2 (Cat # ab211033, Abcam); anti-HO-1 (Cat # EP1391Y, Abcam); anti-GST (Cat # ab138491, Abcam); anti-NQO1 (Cat # 11,451-1-AP, Proteintech, Wuhan, China); anti-FOXO3a (Cat # 66,428-1-Ig, Proteintech); anti-GCLM (Cat # 14,241-1-AP, Proteintech); anti-Keap1 (Cat # 10,503-2-AP, Proteintech). IgG(H + L)(HRP-labeled Goat Anti-Rabbit IgG(H + L)) and IgG(H + L)(HRP-labeled Goat Anti-Mouse IgG(H + L)) were purchased from Beyotime (Beijing, China). All protein expressions were normalized by β-actin expression.

Proteins from cells were extracted and separated by SDS‐PAGE. After transferred onto PVDF membranes (Millipore), the proteins were blocked with 5% skim milk and then incubated with Bax antibody (dilution 1:800; Abcam), cleaved caspase 3 antibody (dilution 1:800; Abcam), Bcl‐2 antibody (dilution 1:800; Abcam), HMGA2 antibody (dilution 1:800; Abcam) or GAPDH antibody (dilution 1:2000; Abcam) at 4℃ overnight. After washed with TBST buffer, the membranes were incubated with HRP‐conjugated secondary antibodies (dilution 1:5000; Beyotime) and the bands were visualized using BeyoECL Moon (Beyotime).

RIPA buffer (Beyotime) was applied for lysing SCC9 and CAL27 cells to isolate total protein. Afterwards, proteins were isolated using 10% SDS-PAGE followed by shifting onto PVDF membranes. Then, 5% skim milk was applied for blocking and primary antibodies were used for incubating membranes overnight at 4 °C. Primary antibodies were listed: anti-CD63 (1:1000, ab134045, Abcam, UK), anti-CD9 (1:1000, ab236630), anti-TSG101(1:1000, ab133586), anti-Calnexin (1:1000, ab133615), anti-p-STAT3 (1:1000, ab76315), anti-STAT3 (1:1000, ab68153), and anti-GAPDH (1:1000, ab9484). Thereafter, membranes were cultivated with goat anti-mouse IgG (HRP) (1:2000, ab6789) for 1 h at 23 °C. BeyoECL Moon (Beyotime) was used for developing and Image J (NIH, USA) was for analyzing bands.

Total protein was extracted from lung tissue and quantified through the BCA assay. Each sample containing 40 μg of mixed loading buffer was boiled at 100 °C for 15 min. The sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with QuickBlock™ blocking buffer (P0235, Beyotime) at 25 °C for 10 min and then rinsed with Western wash buffer (P0023C, Beyotime) for 5 min 3 times. Anti-rabbit p-AMPK (dilution: 1:500, ab133448, Abcam, Cambridge, UK), anti-rabbit AMPK (dilution: 1:1000, ab32047, Abcam, Cambridge, UK), anti-rabbit cleaved-caspase-1 (dilution: 1:500, ab179515, Abcam, Cambridge, UK), anti-rabbit cleaved-caspase-3 (dilution: 1:500, ab32351, Abcam, Cambridge, UK) and GAPDH (dilution 1:1000, K106389P, Solarbio) were used for overnight incubation of PVDF membranes at 4 ℃. After rising with Western washing buffer 3 times, the membranes were incubated with goat anti-rabbit secondary antibody (dilution 1:1000, A0562, Beyotime) at 25 ℃ for 1 h. After washing 3 times with Western washing buffer, protein bands were detected using BeyoECL Moon (P0018, Beyotime). The ratio between the gray value of the target protein and the GAPDH bands (internal reference) was calculated using ImageJ.

Proteins in cells were extracted with precooled radioimmunoprecipitation assay buffer (catalog number: P0013C; Beyotime, Shanghai, China), and protein quantification was performed using a BCA protein concentration determination kit (catalog number: P0012S; Beyotime). Electrophoresis was performed on a 10% sodium dodecyl sulfate-polyacrylamide gel, and the proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (catalog number: IPFL00010; Millipore, Shanghai, China). After incubation with 5% skim milk at 25°C for 2 h, the PVDF membranes containing the proteins were incubated with diluted NEK6 antibody (catalog number: 10378-1-AP; dilution rate: 1 : 1000; Proteintech, Wuhan, China) or GAPDH antibody (catalog number: 10494-1-AP; dilution rate: 1 : 5000; Proteintech) at 25°C for 2 h. After washing, the PVDF membranes were incubated with diluted goat anti-rabbit IgG modified with horseradish peroxidase (catalog number: SA00001-2; dilution rate: 1 : 2000; Proteintech) at 25°C for 1 h. After washing, the PVDF membranes were covered with BeyoECL Moon (catalog number: P0018FM; Beyotime) for exposure. Quantification by densitometry was performed using ImageJ 1.52v (NIH, Bethesda, MD, USA). GAPDH was used as an internal control.