Ab109629

After treatment, the total protein of AF cells from different samples was extracted by using a Western and IP Cell Lysis Kit (Beyotime). Protein concentrations were measured by a BCA Protein Assay Kit (Beyotime). Equal aliquots of protein from each sample were separated by 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes (Millipore). Then, the membranes were blocked by 5% nonfat milk in TBST buffer and incubated overnight at 4°C with primary antibodies, including GAPDH (1:5,000, ab9485; Abcam), Bax (ab32503, 1:1,000; Abcam), Bcl‐2 (ab32124, 1:1,000; Abcam), cytochrome c (Cyt c; ab13575, 1:1,000; Abcam), FoxO1a (ab52857, 1:1,000; Abcam), FoxO3 (ab109629, 1:1,000; Abcam), FoxO4 (ab128908, 1:1,000; Abcam), p‐FoxO1a (S256; ab131339, 1:1,000; Abcam), p‐FoxO1a (S319; ab47328, 1:1,000; Abcam), BIM (ab32158, 1:1,000; Abcam), PI3K (ab151549, 1:1,000; Abcam), AKT (ab170463, 1:1,000; Abcam), and phospho‐AKT (p‐AKT; ab81283, 1:1,000; Abcam). Afterwards, the membranes were incubated with secondary antibodies for 1 hr at room temperature. After washing with PBS three times, the bands were observed in a darkroom and quantified using the ImageJ software.

Protein lysates were separated on 10% SDS-PAGE gels and electrophoretically transferred to PVDF membranes (Millipore, ISEQ00010). Then, the blots were probed with primary antibodies against EGFR (CST, #4405), p-EGFR (CST, #4407), PI3K (Abcam, ab189403), p-PI3K (Abcam, ab182651), AKT (Abcam, ab8932), p-AKT (Abcam, ab179463), FOXO3a (Abcam, ab109629), p-FOXO3a (Abcam, ab154786), p27 (Abcam, ab32034), p21 (Abcam, ab109502), and alpha tubulin (Abcam, ab52866) overnight at 4°C, followed by incubation with an HRP-labeled horse anti-mouse IgG (CST, #7076) or goat anti-rabbit IgG (CST, #7074) for 1 h at room temperature. Signals were detected using enhanced chemiluminescence (ECL). Alpha tubulin was used as the protein loading control.

The cells were treated with the RIPA buffer (Beyotime, Shanghai, China), and the cell lysates were centrifuged at 12000 rpm at 4°C for 10 minutes. The protein concentrations were determined by the BCA kit (Beyotime Inst. Biotech, Beijing, China). Equal amounts of protein (30 μg) were separated on 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk for two hours at room temperature (RT) and incubated with the anti-Akt (Abcam, 1 : 1000, ab8805, Shanghai, China), anti-pAkt (Abcam, 1 : 1000, ab38449), anti-FOXO3a (Abcam, 1 : 1000, ab109629), anti pFOXO3a (Abcam, 1 : 1000, ab154786), anti-NF-κB (Abcam, 1 : 1000, ab32360), anti-pNF-κB (Abcam, 1 : 1000, ab76302), anti-Bcl2 (Abcam, 1 : 1000, ab32124), anti-Bax (Abcam, 1: 1000, ab32503), anti-C-Caspase3 (Abcam, 1 : 1000, ab32042), anti-iNOS (Abcam, 1 : 1000, ab178945), anti-COX2 (Abcam, 1 : 1000, ab62331), and anti-GAPDH (Abcam, 1 : 1000, ab ab9485) antibodies overnight at 4°C. Subsequently, the membranes were washed with TBST four times and incubated at RT with the horseradish peroxidase- (HRP-) labeled anti-rabbit secondary antibody (1 : 3000) for one hour. The protein bands were then visualized using enhanced chemiluminescence (ECL; Pierce; Thermo Fisher Scientific, Inc.). The quantification of bands was performed by using ImageJ software.

Cultured cells were resuspended in RIPA buffer (Beyotime, Jiangsu, China) to isolate the total proteins. A bicinchoninic acid protein assay kit (Beyotime) was used to quantify total protein. Equal amounts of protein were separated on 10% SDS–PAGE gels and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk (w/v) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies against FOXO3 (ab109629; dilution 1:800) or GAPDH (ab181602; dilution 1:1000; both from Abcam, Cambridge, MA, USA). Further, horseradish peroxidase-conjugated secondary antibodies (ab205718; dilution 1:5000; Abcam) were added and incubated for 1 h at room temperature. After washing, an enhanced chemiluminescence reagent (Beyotime) was used for signal production. Protein signals were analyzed utilizing Quantity One software version 4.62 (Bio Rad Laboratories, Inc.).

After the cells and tissues were treated, the culture medium was removed. The protein lysis buffer (Roche) separated the total protein. Then, 50 g total protein went through 2 h of electrophoresis (100 V) on 12% polyacrylamide gel. The protein was transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Moreover, 5% skimmed milk powder sealed the membranes for 1 h (RT). TBST flushed them 3 times (10 min each). The primary antibodies we employed for overnight incubation at 4°C encompassed anti-CCL2 (Abcam, ab214819, 1:1,000, MA, USA), anti-CCR2 (Abcam, ab273050, 1:1,000), anti-NF-κB (Abcam, ab32536, 1:1,000), anti-pNF-κB (Abcam, ab76302, 1:1,000), anti-FOXO3a (Abcam, ab109629, 1:1,000), anti-FOXO3a (Abcam, ab154786, 1:1,000), anti-pFOXO3a (Abcam, ab154786, 1:1,000), anti-Bad (Abcam, ab32445, 1:1,000), anti-Bax (Abcam, ab32503, 1:1,000), anti-cleaved caspase-3 (Abcam, ab13585, 1:1,000), anti-iNOS (Abcam, ab178945, 1:1,000), anti-COX2 (Abcam, ab179800, 1:1,000), and anti-GAPDH (Abcam, ab9485, 1:1,000). Subsequent to a wash in TBST, the anti-rabbit secondary antibody labeled by horseradish peroxidase (concentration: 1:3,000) was given for 1-h incubation at RT. TBST rinsed the membranes 3 times (10 min each). In the end, color and image development was done with the use of the reagent of Western blot (Invitrogen). Image J analyzed each protein's gray value.

Chondrocytes were lysed with immunoprecipitation lysis buffer (Sigma‐Aldrich, MO) to extract total protein. BCA Protein Assay Kit (P0010, Beyotime, China) was used for the quantification of total protein. Protein extracts were subjected to 10% SDS‐PAGE, and transferred to polyvinylidene fluoride membranes. After blockade of 3% BSA, the membranes were incubated with primary antibodies FoxO3 (ab109629, Abcam, UK, 1:1000), COL2A1 (NB‐600‐844, Novus, MO, 1:1000), Sox9 (ab185966, Abcam, UK, 1:5000), ACAN (A11691, ABclonal, China, 1:500), ADAMTS5 (ab41037, Abcam, UK, 1:1000), Runx2 (ab236639, Abcam, UK, 1:1000), Bcl‐2 (ab32124, Abcam, UK, 1:1000), Bax (ab32503, Abcam, UK, 1:1000), PCNA (ab92552, Abcam, UK, 1:1000), and GAPDH (NB100‐56875, Novus, MO, 1:5000) at 4°C for 12 hours, and incubated with HRP goat anti‐rabbit IgG (AS014, ABclonal, China, 1:2000). The membrane was detected using BeyoECL Star (P0018AS, Beyotime, China). GAPDH was used as the internal reference.