Ecl chemiluminescence substrate

The grass carp serum (2ml) was loaded onto a Superdex-200 FPLC column (GE Healthcare) in an ÄKTA purification system (GE Healthcare), then eluted with PBS (pH 7.4) to collect fractions (1 ml per fraction). The fraction with molecular masses of 180-200 kDa was collected to detect C3.1 by Western blot. Briefly, the fractions were resolved by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and the gel was then stained with Coomassie blue dye, followed by transferring to a nitrocellulose blotting membrane (Pall). The membrane was blocked for 1 h at room temperature in TBST buffer (25 mM Tris˗HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) containing 5% nonfat dry milk, then incubated with rabbit anti-grass carp C3a.1 pAbs at 4°C overnight. After three washes with TBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG Abs (Thermo) for 2 h at room temperature. After three washes with TBST, the membrane was stained with ECL chemiluminescence substrate (Biosharp) and photographed by a chemiluminescence imager (Amersham Imager 680, GE Healthcare). The gel slice corresponding to the C3.1 band was subjected to in-gel tryptic digestion, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis.

Total protein samples were extracted from OC cell lines and tissues using RIPA (Cat No. BL504A, Biosharp, China) lysis buffer supplemented with protease/phosphatase inhibitors (Cat No. BL612A, Biosharp, China). Proteins (20 μg/lane) were separated using 10–12% SDS‒PAGE and then transferred onto a PVDF membrane (Cat No. IPVH00010, Millipore, USA). The membrane was then blocked in 5% nonfat milk and incubated overnight with specific primary antibodies at 4 °C. The primary antibodies included anti-β-Actin (1:50,000; Cat No. AC026, ABclonal, Wuhan, China), anti-GAPDH (1:2000; Cat No. 60004-1-Ig, Proteintech, Wuhan, China), anti-CD24 (1:1000; Cat No. AP22227c, Abcepta, Suzhou, China), anti-CD47 (1:1000; Cat No. EM1701-37, HUABIO, Hangzhou, China), anti-HIF-1α (1:2000; Cat No. 79233, CST, USA), anti-p-STAT3 (1:1000; Cat No. 9145, CST, USA), anti-STAT3 (1:1000; Cat No. 4904, CST, USA), anti-Caspase 3/p17/p19 (1:1000; Cat No. 19677-I-AP, Proteintech, Wuhan, China) and anti-C-Myc (1:1000; Cat No. T55150, Abmart, Shanghai, China). After incubation with the corresponding secondary antibodies, bands were detected using ECL chemiluminescence substrate (Cat No. BL520A, Biosharp, China).