| Description |
For immunoprecipitation (IP) assays, protein lysates were prepared from stochastic vacuum fractions (SVFs) from white adipose tissue of mice, 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). |