β actin

DMEM was purchased from BasalMedia. Penicillin/streptomycin and FBS were acquired from Gibco. TRIzol reagent was obtained from Thermofisher. 5x HiScript II Q RT SuperMix and 2x AceQ Universal SYBR qPCR Master Mix were purchased from Vazyme. Recombinant murine M-CSF, IFN-γ, and IL-4 protein were purchased from Peprotech. The antibodies are listed as follows: p-STAT1 (Abclonal), STAT1 (Abclonal), p-STAT3 (Abclonal), STAT3 (Cell Signaling Technology, CST), p-STAT6 (Abcam), STAT6 (Abclonal), p-ERK (CST), ERK (CST), p-JNK (CST), JNK (CST), p-P65 (CST), P65 (CST), p-P38 (CST), P38 (Abclonal), iNOS (CST), Arg1 (CST), PRMT2 (Novus), β-actin (CST), PE anti-mouse F4/80 (Biolegend), FITC anti-mouse CD86 (Biolegend), APC anti-mouse CD206 (Biolegend).

Protein from tissues and cells was extracted in RIPA buffer (Solarbio, Beijing, China). Protein concentrations were determined by the bicinchoninic acid method. Western blotting was used to detect protein expression. One hundred microgram of protein extract was separated by SDS‐PAGE (Beyotime) and transferred to PVDF membranes, which were blocked with 5% non‐fat milk for 1 hr. Membranes were incubated with primary antibodies overnight at 4°C. After washing three times in 1× Tris‐buffered saline with Tween‐20, membranes were incubated with secondary antibodies (Zsgb‐bio, Beijing, China) for 1 hr. Relative band intensities were evaluated using Quantity One (Bio‐Rad MP4000, Hercules, CA, USA). Primary antibodies were calponin and α‐SMA (1:500; Millipore), KLF5 (1:50; Abcam, Cambridge, MA, USA), MYOCD (1:200; Abcam) and β‐actin (1:500; Biolegend, San Diego, CA, USA).

Western blot analysis was performed as previously described [37 (link)]. Cytoplasmic and nuclear extracts were prepared using a Nuclear Extract kit (Active Motif) according to manufacturer's instructions. Protein concentration was measured by a BCA protein assay kit. Samples were mixed with 6×SDS reducing sample buffer and boiled for 10 minutes before loading. Equal protein concentrations (20μg/lane) were separated on an SDS polyacrylamide gel and transferred electronically to PVDF membranes. The membranes were blocked with 5% nonfat milk in TTBS (50 mM Tris [pH 7.5], 0.9% NaCl, and 0.1% Tween-20) for 1 hour at room temperature and incubated with primary antibodies to dectin-1 (1:200), PGLYRP-2 (1:200), or β-actin (622102, BioLegend, 1:1000) overnight at 4°C. After three washes with Tris-buffered saline with 0.05% Tween 20 for 10 min each, the membranes were incubated with HRP conjugated rabbit anti-goat IgG (1:1000) or goat anti-rabbit IgG (1:1000, Rockford, IL) for 1h at room temperature. The signals were detected with an ECL Plus chemiluminescence reagent (GE Healthcare), and the images were acquired by a blot imaging station (model 2000R; Eastman Kodak, Rochester, NY).

Whole-cell lysates were resolved by SDS-PAGE (40 μg of protein was loaded into each well) using 10% acrylamide mini-gels, followed by electrophoretic transfer to PVDF membranes (Immobilon-P MILLIPORE) for 2 h. Membranes were blocked with 5% fat-free milk in PBS for 2 h and incubated with primary antibodies overnight. The detection step was performed with peroxidase-coupled anti-mouse IgG and anti-rabbit IgG (BioLegend, 1 : 2000) and anti-goat IgG (Santa Cruz Biotechnology) for 2 h. The primary antibodies included antiphosphotyrosine (Santa Cruz Biotechnology) and β-Actin (BioLegend). All primary antibodies were diluted 1 : 500 in 1% fat-free milk in PBS. Blots were developed with the ECL detection system according to the manufacturer's instructions (Amersham). Blots are representative of two separate experiments.

Cell lysates and tissue lysates were harvested with RIPA buffer (Biosesang, Seongnam, Republic of Korea) containing a protease inhibitor cocktail and a phosphatase inhibitor. The proteins were fractionated on 10% SDS-polyacrylamide gels. The gels were transferred to polyvinylidene fluoride membranes, and the membranes were incubated for 1 h in 5% skim milk in TBS-T buffer. Then, the membranes were incubated with primary antibodies recognizing pAkt (cat#2965), pSmad2/3 (cat#8828), ColIα1 (cat#84336) (Cell Signaling Technology, Danvers, MA, USA), Akt (cat#610861), Smad2/3 (cat#610843) (BD Biosciences, CA, USA), and β-actin (cat#664802, BioLegend, San Diego, CA, USA), followed by incubation with an HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Signals were developed using an enhanced chemiluminescence system (Thermo Fisher Scientific). The optical densities of the target protein bands were analyzed with ImageJ 1.52a (National Institutes of Health, Bethesda, MD, USA).