Phenylmethylsulfonyl fluoride pmsf

The Hrd1 protein level was determined by using Western blot analysis as previously described.21 (link) Briefly, the tissues or cells were dissociated on ice and homogenized in a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing 50 μL/mL protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Beyotime). The protein concentration in the supernatants was determined by the BCA method. Samples containing 20 µg of protein were boiled and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% Tris-glycine gels and electrophoretically transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% fat-free milk in Tris-buffered solution containing 0.05% Tween-20 for 1 hour at room temperature and then incubated with rabbit anti-human Hrd1 (1:5,000; Santa Cruz Biotechnology) overnight at 4°C. The membrane was washed and incubated with a horseradish peroxidase-linked secondary antibody (1:2,000, Beyotime) and finally processed by using an ECL chemiluminescence reaction kit (Millipore, Billerica, MA, USA), followed by exposure to medical film. The band density of the target protein relative to that of β-actin was quantified with Bio-Rad Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA, USA).

Phorbol-12-myristate-13-acetate (PMA) was bought from Sigma Aldrich (St. Louis, Missouri, USA), human IL-4 and human IL-13 were purchased from Peprotech (200-04 and 200-13, Rocky Hill, USA).The anti-CD163, anti-CD206, anti-β2-AR and anti-PKA antibodies were purchased from Santa Cruz Biotechnology(sc-33559, sc-58986, sc-271322 and sc-98951, Ca, USA). Phenylmethylsulfonyl fluoride (PMSF) was bought from beyotime biotechnology (Shanghai, China). Primary antibodies against STAT3, pSTAT3, pCREB were purchased from Cell Signaling Technology (4904s, 9131s and 9198s, Danvers, MA, USA). Anti-CREB antibody was purchased from Abcam (ab32515, Cambridge, MA, USA). AlexaFluor488 donkey anti-mouse antibody and AlexaFluor594 goat anti-rabbit antibody were purchased from ThermoFisher Scientific (A11037, A21202, Waltham, MA, USA). Fluorescein isothiocyanate dextran (FITC-dextran) was from Sigma Aldrich (FD40s, St. Louis, Missouri, USA). AlexFluor 647 was from Fcmacs (FMS-Msaf64701, Nanjing, Jiangsu, China).

Rabbit anti-human anti-BMP-7 antibody was obtained from Abcam, Cambridge, UK (cat. no. ab56023). Rabbit anti-human anti-gremlin antibody was purchased from Wuhan Boster Biological Technology, Ltd., Wuhan, China (cat. no. BA2287). Rabbit anti-human anti-p-Smad1/5/8 antibody was purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA (cat. no. sc-12353-R). The 3,3′-diaminobenzidine tetrahydrochloride (DAB) kit was obtained from ZSGB-Bio, Beijing, China. RNAstore solution, TIANScript first-strand cDNA synthesis kit, and SuperReal PreMix Plus SYBR Green were obtained from Tiangen Biotech Co., Ltd., Beijing, China. RIPA lysis buffer and phenylmethylsulfonyl fluoride (PMSF) were obtained from Beyotime Institute of Biotechnology, (Haimen, China).