Hrp labeled anti mouse igg

Western blot analysis was performed using anti-DYKDDDDK-tag antibody and anti-α-tubulin antibody. The cell lysate was mixed with 2× Sample buffer (2% SDS, 0.1 M Tris-HCl pH 7.5, 25% glycerol, appropriate amount of BPB), boiled at 95°C for 3 min, allowed to stand on ice for 5 min, and then subjected to SDS-PAGE. After electrophoresis, proteins in the gel were transferred to the Hybond P membrane (GE Healthcare) at 15 V for 30 min. The FLAG-PLAT2 was detected with anti-DYKDDDDK tag polyclonal antibody (Cell Signaling Technology) as a primary antibody, and HRP-labeled anti-rabbit-IgG (Cell Signaling Technology) as a secondary antibody. After incubation, chemiluminescence was detected using Luminata Forte Western HRP Substrate (Millipore) and Ez-Capture II (ATTO). To strip the antibody, the membrane was incubated for 30 min at 60°C using stripping buffer (50 mM Tris-HCl, pH 6.8, 2%SDS, 100 mM β-mercaptoethanol). The α-tubulin was detected with anti-α-tubulin antibody (MBL) as a primary antibody and HRP-labeled anti-mouse-IgG (Cell Signaling Technology) as a secondary antibody.

Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (R0010, Solarbio Science & Technology Co., Ltd., Beijing, China) containing PMSF, and protein contents were measured using the Micro BCA Protein Assay Kit (Thermo Fisher). The lysates were centrifuged and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, electrotransferred onto a polyvinylidene fluoride membrane. The membranes were blocked and incubated with the specific primary antibody overnight at 4°C. After washing with TBST, the membrane was subjected to incubation with horseradish peroxidase- (HRP-) labeled anti-rabbit IgG (1 : 1000, #7074, Cell Signaling Technology, Beverly, MA) or HRP-labeled anti-mouse IgG (1 : 1000, #7076, Cell Signaling Technology) for 2 h. The enhanced chemiluminescence reagents (Amersham, Piscataway, NJ) and ChemiDoc XRS system (Bio-Rad, Hercules, CA) were adopted to visualize protein expression. The primary antibodies were as follows: anti-NRF2 (1 : 1000, #ab92946, Abcam), anti-Col II (1 : 400, #ab34712, Abcam), anti-Aggrecan (1 : 1000, #ab36861, Abcam), anti-ADAMTS-5 (1 : 250, #ab41037, Abcam), anti-MMP13 (1 : 500, #ab39012, Abcam), anti-ACSL4 (1 : 10000, # ab155282, Abcam), anti-GPX4 (1 : 1000, #ab125066, Abcam), anti-FTL (1 : 10000, #ab109373, Abcam), and anti-β-actin (1 : 2000, #4967, Cell Signaling Technology).

RAW cells were lysed in CelLyticM lysis buffer (Sigma-Aldrich) with protease and phosphate inhibitor cocktails (Nacalai Tesque, Kyoto, Japan). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories Hercules, CA, USA), transferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories), and probed with an anti-mouse p44/42 MAPK (Erk1/2) rabbit mAb, anti-mouse phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP rabbit mAb, anti-mouse SAPK/JNK rabbit mAb, anti-mouse phospho-SAPK/JNK (Thr183/Tyr185) (81E11) rabbit mAb, anti-mouse p38 MAPK rabbit monoclonal antibody (mAb), anti-mouse phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP rabbit mAb, anti-mouse-NF-κB p65 (D14E12) XP rabbit mAb, anti-mouse phospho-NF-κB p65 (Ser536) (93H1) rabbit mAb, or anti-mouse β-actin (8H10D10) mouse mAb (all purchased from Cell Signaling Technologies, Danvers, MA, USA). Horseradish peroxidase-labeled anti-rabbit IgG, HRP-labeled anti-mouse IgG (Cell Signaling Technologies), and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) were used as secondary antibodies. ImmunoStar LD (WAKO, Tokyo, Japan) and the RAS-3000 System were used to visualize the proteins.