Phenylmethanesulfonyl fluoride pmsf

The fibrinolytic enzyme type was determined by evaluating the effects of multifarious inhibitors on the fibrinolytic activity. Briefly, the purified enzyme solution (50 IU/ml) was pre-incubated at 37 °C for 30 min separately with each of the following inhibitors: 5.0 mM serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF; Sigma, Beijing, China), 5.0 mM metalloprotease inhibitor ethylene diamine tetraacetic acid (EDTA; Sigma, Beijing, China), 10 µM aspartic protease inhibitor pepstatin A (Sigma, Beijing, China), 1.0 mM cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64; Sigma, Beijing, China), and 1.0 mM aminopeptidase inhibitor bestatin (Sigma, Beijing, China). To measure the effects of metal ions on the activity, the fibrinolytic enzyme (50 IU/ml) was pre-incubated separately with different metal ions (final concentration 5 mM) at pH 7.5 and 37 °C for 30 min. The enzyme solution without added ions was used as a control.

Whole-cell lysates from iEPCs were prepared using radioimmunoprecipitation assay (RIPA) buffer (150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8) containing Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL, USA) and phenylmethanesulfonyl fluoride (PMSF) (Sigma Aldrich, St. Louis, MO, USA). The lysates were quantitated using the Bradford Reagent (Biorad Inc., Richmond, CA, USA). A total of 30 μg of the lysate was loaded on a 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and analyzed by western blot using primary antibodies, anti-BCL11A (1:1000 dilution) (Cell Signaling Technologies, Danvers, MA, USA), and anti-actin (1:5000 dilution) (BD Pharmingen, San Jose, CA, USA) and secondary antibodies, anti-mouse IgG HRP (Cell Signaling Technologies, Danvers, MA, USA) and anti-rabbit IgG HRP (Invitrogen Corporation, Camarillo, CA, USA). The signal was detected using the Westar Supernova (Cyanagen, Bologna, Italy) and FluorChemE gel documentation system (Protein Simple, San Jose, CA, USA).

Lipid rafts from RAW 264.7 cells were isolated as described previously [25 (link),26 (link)]. Briefly, cells were washed with ice-cold PBS, scraped in 2 mM EDTA/PBS, centrifuged (500× g, 5 min, 4 °C), and incubated on ice for 30 min in 600 μL lysis buffer (150 mM NaCl; 25 mM MES; 5 mM EDTA, pH 6.5; 1% Triton X-100) supplemented with a cocktail of protease inhibitors (Sigma-Aldrich, Burlington, MA, USA) and phenylmethanesulfonylfluoride (PMSF; Sigma-Aldrich, Burlington, MA, USA). Samples were homogenized by passing through a 25⅝ G needle. The homogenate was centrifuged (8000× g, 5 min, 4 °C) and supernatant was adjusted to a final concentration of 40% (w/v) iodixanol (OptiPrep^®, Sigma-Aldrich, Burlington, MA, USA), and the mixture was then layered under a 20–40% discontinuous iodixanol gradient and centrifuged at 260,000× g for 16 h at 4 °C using an SW 41 Ti Rotor (Beckman Coulter, Brea, CA, USA). Then, 11 fractions of 1 mL were collected from the top to the bottom of the gradient tube, and Western blot analysis of the fractions was performed. Lipid raft fractions were defined by the presence of a raft marker, flotilin 2 [27 (link)], while TfR1 was considered as a marker of non-lipid raft domains [28 (link)].