Anti tubulin

Western blot experiments were performed as previously [67 (link)]. Anti-SRGN (1:100, sc-374657, Santa Cruz Biotechnology), Anti-HIST1H1C (1:1000, ab4086, abcam), Anti-APOE (1:1000, #13366, Cell Signaling Technology), anti-Tubulin (1:5000, #T0023, Affinity Biosciences) and anti-β-Actin (1:4000, #T0022, Affinity Biosciences) were used as primary antibodies. The blots were imaged with the ChemiScope System (Clinx, China).

Membrane and Cytosol Protein Extraction Kit (Beyotime, China) and Bicinchoninic Acid Protein Assay Kit (Beyotime) were used for protein extracted and quantified. Proteins were separated on 10% SDS polyacrylamide gel (Beyotime) and then blotted onto PVDF membranes (Millipore, USA). The PVDF membranes were incubated in 5% skim milk for 2 h. After that, all membranes were cultured with the primary antibodies of anti-Tubulin (1:10,000, Affinity, USA), anti-VEGF-A (1:1000, Affinity), anti-Pi3k (1:1000, Affinity), anti-P-Pi3k (1:1000, Affinity), anti-Akt (1:1000, Affinity) and anti-P-Akt (1:1000, Affinity) overnight at 4 °C. The membranes were then incubated with corresponding secondary antibody (1:3000, Affinity) for 1 h. The protein bands were finally detected by chemiluminescence detection system (ProteinSimple, USA).

Total protein was homogenized and extracted from 3 pooled embryos at 3 dpf using radioimmunoprecipitation assay (RIPA) solution (Beyotime Biotechnology, Shanghai, China) and was subjected to standard western blotting analysis. Briefly, extracted total protein was separated in 6% polyacrylamide gels, transferred onto nitrocellulose membrane, and subsequently incubated with primary and secondary antibodies. The primary antibody for anti-vmhc was obtained from proteintech (1:1,000, 22280-1-AP), and anti-tubulin was purchased from Affinity Biosciences (1:2,000, T0034). The secondary antibody was purchased from Affinity Biosciences (S0002). Quantification of relative protein expression was performed using Image J and student t-test was used to calculate the P-value.