Tca protein precipitation kit

Transfected HEK293 cells and hFOB1.19 cells were lysed using radio immunoprecipitation assay (RIPA) buffer containing the protease inhibitor and PMSF. Secreted proteins in FBS‐free DMEM supernatants of HEK293 cells were concentrated using the TCA Protein Precipitation Kit (Sangon Biotech) and redissolved in 1 × denature protein loading buffer. Proteins were separated through 12% SDS–polyacrylamide gels and transferred to 0.22 μm nitrocellulose filter membranes. A rabbit anti‐PHEX polyclonal antibody (Sigma‐Aldrich, SAB2700899) was used at a 1:2000 dilution, and a rabbit anti‐ FGF23 polyclonal antibody (Abclonal, A6124) was used at a 1:1,000 dilution. Secondary HRP‐conjugated goat anti‐rabbit antibody (Sangon Biotech) was used at a 1:10,000 dilution. The signals of antibody immune complexes were captured by the FUSION Solo S imaging system (Vilber Lourmat) using the BeyoECL Star chemiluminescence Kit (Beyotime Biotech).

The TCA Protein Precipitation Kit (Sangon Biotech, China) was used to separate protein pellets from cellular supernatant in different HOXA10 expression cells according to the manufacturers’ instructions. The protein extracts from TCA assays were then used for western blot detection.

S-Nitrosoglutathione (GSNO, N4148), DTT (D0632), and Puromycin (P8833) were from Sigma (St. Louis, MO). L-NAME HCl (S2877) and Cycloheximide (CHX, S7418) were purchased from Selleck Chemicals (Houston, TX). Cavosonstat (N91115) were from STA for Nivalis (Shanghai). TCA Protein Precipitation Kit was purchased from Sangon Biotech (Shanghai, China). Antibodies against VEGFD(C-12), FLK1 (A-1), PECAM-1 (H-3), and c-Myc were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against VEGF receptor 3 (VEGFR3) were from Cell Signaling Technology (Danvers, MA). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), HA, VEGFD, and LYVE1 were from HUABIO (Hangzhou, China). The IRDye 680 and 800 secondary antibodies were from LI-COR Bioscience (Lincoln, Nebraska). Alexa555- and Alexa488-conjugated antibodies were from Molecular Probes (Eugene, OR).