Bradford protein assay kit

CFs were washed with PBS (0.1 mol/L, pH 7-7.4) three times, lysed in a lysis buffer for 30 min at 4 °C, and then scraped off using a cell scraper before being mixed with a one-quarter volume of 5× loading buffer. The cell protein mixture was then boiled in a 100 °C water bath and cooled to room temperature. The protein concentration was determined using a Bradford protein assay kit (Amresco, M173-KIT). Equal amounts of protein (50 µg) were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The strips were blocked with 5% nonfat milk for 1 h at room temperature. The strips were then incubated overnight with primary antibodies against AAT1 (Sigma, AV48205, 1:500), collagen I (Abcam, ab254113, 1:500), collagen III (Abcam, ab7778, 1:500), α-SMA (Abcam, ab5694, 1:500), β-catenin (Abcam, ab6302, 1:500), p-P38 MAPK (Abcam, ab4822, 1:500), P38 MAPK (Abcam, ab170099, 1:500) and GAPDH (Abcam, ab181602, 1:2000) diluted in PBS with 0.05% Tween 20 at 4 °C. The strips were rinsed using PBS with 0.05% Tween 20 for 30 min, followed by incubation with secondary antibodies for 1 h at room temperature. All protein bands were detected using Amersham ECL Western blotting detection reagents (GE Healthcare, RPN2106) and analyzed with a biological electrophoresis image analysis system.

The protein obtained from the various experiments was isolated using 1 × cell lysis buffer (Cell Signaling, Danvers, MA, USA) and the concentration was determined with the Bradford Protein Assay Kit (AMRESCO, Solon, OH, USA). Protein lysates were then investigated by Western blot analyses as previously described [39 (link),42 (link)]. The specific antibodies used in the current study were listed as follows: CP (abcam, Cambridge, UK), Fibrinogen, SOD and β-actin. The band intensity was quantified by using GeneTools software and the level of β-actin was performed as internal control.

Western blot analysis was applied to perform and quantify the amount of protein. The protein obtained from the skin was isolated using 1× cell lysis buffer (Cell Signaling, Danvers, MA, USA) and the concentration was determined with the Bradford Protein Assay Kit (AMRESCO, Solon, OH, USA). The specific antibodies used in the current study were listed as follows: α-SMA (Santa Cruz, Dallas, TX, USA, sc-32251), COLA1 (Santa Cruz, sc-8784), CD68 (Santa Cruz, sc-20060) and GAPDH (Santa Cruz, sc-25778), N-cadherin (Epitomics, Burlingame, CA, USA, 2019-1), ZO-1 (Cell Signaling, 9782), phospho-ERK (Cell Signaling, 9101) and ERK (Cell Signaling, 4695), activin A (myBioSource, San Diego, CA, USA, MBS7103066 & MBS9201920), and CD163 (Bioss, Woburn, MA, USA, bs-2527R). The band intensity was quantified by using GeneTools software (Syngene, Cambridge, UK) and the level of GAPDH was performed as internal control [44 (link)]. All experiments were performed in biological triplicate to confirm the reproducibility.