| Description |
For immunoprecipitation (IP) assays, protein lysates were prepared from stochastic vacuum fractions (SVFs), using Pierce TM IP Lysis Buffer (Thermo, 87787). The lysates were incubated overnight at 4 centigrade with anti-ANXA1(Abcam ab214486, 1:100), anti-PDLIM7 (Proteintech 10221-1-AP, 1:100), or control immunoglobulin G (IgG; Beyotime, A7058) on a rotating pattern. Immobilized Protein A/G resin slurry (Thermo, 20423) was added to the lysates and incubated for 2 hours at 4 centigrade with gentle mixing. The complexes were washed five times with lysis buffer (Thermo, 28379) and resuspended in 2-SDS loading buffer. The immunoprecipitated proteins were eluted by incubation at 95°C for 5 minutes and detected by immunoblotting after separation by SDS-PAGE.For mass spectrometry-based proteomic analysis, gel pieces were dehydrated with acetonitrile and digested with trypsin. LC-MS/MS analysis of peptides was conducted at Peking University Institute of Systems Biomedicine (China). |
