Micro bca protein assay kit

At the end of reoxygenation, adherent cardiomyocytes were washed twice with PBS and 1 mL of strong RIPA cell lysate (strong RIPA cell lysate: PMSF, 100:1), then centrifuged (12,000 g at 4°C) for 5 min. The supernatant was aliquoted, and protein content was determined using the Micro BCA protein assay kit (Beyotime). After denaturation at 100°C, proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Amresco, Solon, OH, USA). The fractionated products were electrophoretically transferred onto a polyvinylidene difluoride membrane (Hybond-P, Bio-Rad, Berkeley, CA, USA) and blocked at 37°C for 2 h. The membrane was then incubated overnight with primary antibodies. HIF-1α, VEGF, Bcl-2, and Bax were probed with 1:1,000 dilutions of the respective antibodies overnight at 4°C. The membrane was then washed with TBST buffer and incubated with secondary antibodies (1:10,000) at room temperature for 2 h. The fluorescence intensity of each membrane was measured by an infrared fluorescent imaging system (NatureGene Corp, Beijing, China) to determine the fluorescence intensity of the target membrane.

Goldfish MCSF-2 ORF excluding singnal peptide was amplified by PCR using gene-specific primers that included 5’-end BamH I and Hind III insertions (Table S1). Restriction enzymes BamH I and Hind III were used to thoroughly digest the PCR result. (Thermo Fisher Scientific, USA), then ligated to the pET32a (+) vector digested with BamH I/Hind III. This resulted in the creation of the recombinant plasmid pET32a-gfMCSF-2, which were subsequently converted into competent E. coli (BL21/DE3) cells (TransGen, China). To express the RgMCSF-2 proteins, 0.1 mM IPTG induction was carried out at 16°C overnight, and recombinants proteins expression was analyzed by SDS-PAGE. Purification of the recombinant protein was accomplished by using Ni-NTA Sefinose Resin (Sangon, China) as directed by the manufacturer. As a control protein in subsequent experiments, the pET32a (+) vector containing a thioredoxin tag without an insert was expressed and purified in the same manner. Subsequently, a ProteoSpin Endotoxin removal column (Norgen Biotek, USA) was used to purify the recombinant protein after it was dialyzed overnight at 4°C in 1 x PBS. Micro BCA Protein Assay Kit (Beyotime, China) was used to determine protein content.

After quantification of the protein samples using a Micro BCA™ Protein Assay Kit (Beyotime, Shanghai, China), equivalent amounts of protein (30 µg) were electrophoresed on 12.5% SDS–PAGE gels and then electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking, the membranes were incubated at 4 °C overnight with the following primary antibodies: GAPDH (1:1000), EGF-C (1:1000), VEGFR-3 (1:1000), AKT (1:1000), p-AKT (1:1000), ERK1/2 (1:1000), p-ERK1/2 (1:1000), IκBα (1:1000), p-IκBα (1:500), NF-κB p65 (1:1000), and p-NF-κB p65 (1:000) and then incubated with the aforementioned anti-mouse or anti-rabbit secondary antibodies (1:2000) at 37 °C for 1 h. Finally, chromogenic results of the target protein were visualized with an Odyssey infrared laser imaging system (Licor, Lincoln, NE, USA). VEGF-C and VEGFR-3 antibodies were purchased from Abcam (Cambridge, UK), and the other antibodies were purchased from CST (Danvers, MA, USA). The intensity of the selected bands was quantified and analysed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).