Ecl plus substrate

Tissues were homogenized in mammalian tissue protein extraction reagent (KeyGEN, Nanjing, China) supplemented with protease inhibitor and phosphatase inhibitor. For western immunoblotting, 25 to 60 μg of protein was separated by 10–15% Bis-Tris protein gel (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and blocked with 5% nonfat milk or 1% bovine serum albumin in tris-buffered saline tween-20 for 2 h at room temperature. The membranes were then incubated overnight with antibodies against p66shc, phospho-p66shc, foxo3a, manganese superoxide dismutase (MnSOD) (Abcam Ltd., Cambridge, UK); phospho-foxo3a (Beyotime Institute of Biotechnology, Hangzhou, China); NF-κB (Santa Cruz Biotechnology, CA, USA); Bcl-xL (Proteintech group, Wuhan, China); cleaved-caspase-3 (Bioworld, Minneapolis, MN, USA); Histone H3.1 (Santa Cruz Biotechnology); or β-actin (Beyotime Institute of Biotechnology). After incubation with appropriate horseradish peroxidase-conjugated secondary antibodies, membranes were developed with ECL plus substrate (Beyotime Institute of Biotechnology). Images were documented with a BioSpectrum-410 multispectral imaging system with a Chemi HR camera 410 (UVP, Upland, CA, USA) and quantitated with the Gel-Pro Analyzer Version 4.0 (Media Cybernetics, Rockville, MD, USA).

The sample solution containing the target protein was obtained by centrifugation, ultrasonic fragmentation and centrifugation. The target proteins were purified by affinity chromatography using Ni Focurose 6FF (IMAC) (HUIYAN Bio, China). After overnight dialysis, the purified protein was filtered and concentrated by 100 kDa Amicon Ultra-15 centrifugal filters (Merck Millipore Ltd). The final concentration of the protein was determined by a BCA protein assay kit (Beyotime, China). Finally, four target proteins denoted ferritin, YaH3F, YaH4F, and YaH5F were obtained and stored at − 80 °C.
The expression of all proteins was verified by using reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified protein was used to verify the effect of ferritin self-assembly through 6% nonreducing SDS–PAGE, followed by blotting and then analysis by Western blotting using an anti-His tag monoclonal antibody (Invitrogen, Carlsbad, CA, USA). Western blotting was performed by using the ECL Plus substrate (Beyotime Biotech, Inc., China).

Total protein was extracted via RIPA buffer, which was evaluated by BCA kit (Beyotime). Samples were separated by SDS-PAGE and transferred onto PVDF membrane. Following being blocked, membrane was incubated by anti-C3AR1 (1:1,000; ab126250; Abcam, USA) or anti-GAPDH (1:1,000; ab8245) at 4°C overnight, followed by being probed with secondary antibody (1:5,000; ab7090). The protein bands were then visualized through ECL Plus substrate (Beyotime).