Anti cox2

Radio‐immune precipitation assay lysis buffer (Beyotime, P0013B) was used for tissue and cell lysates. BCA assay kit (Beyotime, P0012S) was used for protein concentration determination. After 30 µg protein amount was separated using 4%–20% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, it was transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk at 37°C for 2 h and then incubated with primary antibodies at 4°C overnight and secondary antibodies at 37°C for 1 h. After washing, the membranes were visualized by an ECL Kit (MedChemExpres) and quantified with ImageJ software. All experiments were repeated in triplicate for statistics. Following primary antibodies were used: anti‐iNOS (1:1000, Proteintech), anti‐COX‐2 (1:1000, Proteintech), anti‐IL‐6 (1:1000, Proteintech), anti‐TLR4 (1:1000, Proteintech), anti‐MYD88 (1:1000, Proteintech), anti‐NF‐κB‐p65 (1:1000, Affinity), anti‐NF‐κB‐p‐p65 (1:1000, Affinity), anti‐CD74 (1:1000, Abcam), β‐Actin (1:2000, Abcam).

In a solution containing a protease inhibitor (AS1008; Aspen), PC12 cells were dissolved on ice for 30 min. BCA protein assay kit was used to measure the protein concentration (PC0020; Solarbio). The protein samples were heated at 100°C for 10 min to completely denature the proteins. The protein samples were then separated by SDS‐PAGE electrophoresis and transferred to PVDF membranes (IPVH00010; Millipore). After being blocked for an hour with 5 percent skim milk, the membrane was incubated with the primary antibody at 4°C overnight. The antibodies used in this study consisted of anti‐DHODH (1:1000, 14877‐1‐AP; Proteintech), anti‐ACSL4 (1:1000, 22401‐1‐AP; Proteintech), anti‐COX2 (1:1000, 66351‐1‐Ig; Proteintech), anti‐GPX4 (1:1000, 67763‐1‐Ig; Proteintech), anti‐ALOX15 (1:1000, ab244205; Abcam), anti‐P53 (1:1000, AB_297667; Abcam). After being washed three times with TBST, the membrane was incubated for 10 min at room temperature with a secondary antibody that had been enzyme‐labeled. Imaging was carried out using an ECL chemiluminescence substrate (BL520B; Biosharp). Band intensities were quantified using Image J software.

Whole cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA).¹⁹ (link) Immunoblotting was performed with anti-MUC1-C (#MA5-11202, 1:100; Thermo Fisher Scientific), anti-ZEB1 (#3396, 1:1000 dilution; Cell Signaling Technology (CST), Danvers, MA, USA), anti-TWIST1 (ab50887, 1:1000 dilution; Abcam, Waltham, MA, USA), anti-BMI1 (#6964, 1:1000 dilution; CST), anti-MYC (#ab32072, 1:1000 dilution; Abcam), anti-SOX2 (#3579, 1:1000 dilution; CST), anti-GLUT1 (#ab115730, 1:100000 dilution; Abcam), anti-HK2 (#2867, 1:1000 dilution; CST), anti-SDHD (#NBP2-83505, 1:1000, NOVUS Biologicals, Centennial, CO, USA), anti-cytochrome c (#10993, 1:4000 dilution; Proteintech; Rosemont, IL, USA), anti-ND1 (#19703, 1:1000 dilution; Proteintech), anti-COX2 (#55070, 1:5000 dilution; Proteintech), anti-TFAM (#8076, 1:1000 dilution; CST), anti-TFB2M (#24411, 1:500 dilution; Proteintech), anti-mTERF3 (#ab230232, 1:1000 dilution; Abcam), anti-SOD2 (#13141, 1:1000 dilution; CST), anti-PRDX3 (#10664, 1:2000 dilution; Proteintech), and anti-β-actin (A5441; 1:50000 dilution; Sigma, St. Louis, MO, USA).

Cells were harvested by 0.25% trypsin (Gbico), cell suspension was then centrifuged, pellet cells were resuspended with 1× sample buffer, and boiled for 12 min. The protein samples were separated by SDS-PAGE and transferred into 0.45 μm PVDF membranes, 2 h later, membranes were blocked by 5% non-fat milk for 1 h, then incubated with primary antibody 30 min-2h in 5% non-fat milk followed by incubation with 1:5000 HRP secondary antibodies for 1 h. Detection of immunoreactive protein on Fuji x-ray film using ECL chemiluminescent substrate (Bio-Red). The following primary antibodies were used: anti-GAPDH and anti-Beta-Tubulin were obtained from Santa Cruz Biotechnology; anti-COX4, anti-COX2, anti-ATAD3A, anti-Mic60, anti-Yme1L, and anti-Tom20 were from Proteintech; anti-OPA1 was obtained from BD Biosciences, anti-Sam50 was obtained from Abcam; anti-Mic19 was from Abclonal; anti-Mic10 was from Invitrogen.

Proteins were extracted from cultured HESCs using RIPA lysis buffer, and the concentration of protein was detected by the BCA Protein Assay Kit (Beyotime). Samples were then separated by 10% SDS-PAGE gels (Beyotime). The primary antibodies were applied according to the provided recommendations: anti-ILK (1:5000, Abcam), anti-TGFβ1 (1:200, Boster), anti-SMAD2 (1:1000, Affinity), anti-VEGF (1:200, Boster), anti-COX-2 (1:700, Proteintech Group), anti-MMP-9 (1:800, Abcam) and anti-GAPDH (1:1000, Abcam). Finally, positive bands were detected using the chemiluminescent ECL Plus reagent (Beyotime) according to the manufacturer’s protocol. The densitometry of bands was quantified using Quantity One version 4.6.0 software (Bio-Rad). Expressions of proteins were normalized to GAPDH protein.

The whole cell protein was extracted with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China, Cat#S711-001S), and the protein concentration was measured with BCA protein analysis kit (Beyotime Biotechnology, Cat#P0012). Equal amounts of total protein were loaded on 6%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the separated proteins were transferred to PVDF membrane (Merck Millipore, Darmstadt, Germany, Cat#IPVH00010). After blocking with 5% skim milk, membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with the IRDye conjugated secondary antibody for 1 h at room temperature. The antibodies used in this study were as follows: Anti-β-Actin (1:1000, Abcam, Cambridge, MA, USA, Cat#ab97626), Anti-GOLPH3 (1:1000, Abcam, Cat#ab91492), Anti-COX-2 (1:1000, Proteintech, WuHan, China, Cat#27308-1-AP), Anti-iNOS (1:1000, Proteintech, Cat#18985-1-AP) and IRDye-conjugated (680RD Goat anti-Mouse/800CW Goat anti-Rabbit) IgG secondary antibodies (1:10000, Li-COR Biosciences, Lincoln, NE, USA, Cat#926-68070/Cat#926-32211). Membranes were imaged with an Odyssey CLX Infrared Imaging System (Li-COR Biosciences).