Ab20346

The cells or exosomes were lysed with RIPA lysate (Sigma) to obtain proteins. The BCA Assay kit (Solarbio, Beijing, China) was used to determine the protein concentration. An equal amount of protein was added onto 10% SDS-PAGE gels, and the protein was then transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The blots were then incubated with primary antibodies, Anti-PI3Kγ antibody (1 : 1,000, ab32089, Abcam), Anti-CD163 antibody (1 : 1,000, ab213612, Abcam), Anti-CD206 antibody (1 : 1,000, sc-58986, Santa Cruz Biotechnology), Anti-PTEN antibody (1 : 1,000, ab267787, Abcam), Anti-AKT antibody (1 : 1,000, ab213612, Abcam), Anti-pAKT antibody (1 : 1,000, ab38449, Abcam), Anti-Vimentin antibody (1 : 1,000, ab20346, Abcam), Anti-α-SMA antibody (1 : 1,000, ab108424, Abcam), Anti-IL-10 antibody (0.5 µg/ml, ab134742, Abcam), Anti-CD63 antibody (1 µg/ml, ab38418, Abcam), Anti-CD61 antibody (1 : 1,000, ab119992, Abcam), Anti-HSP70 antibody (1 : 1,000, ab2787, Abcam), and anti-Actin (1 µg/ml, ab8286, Abcam) overnight at 4°C. The horseradish peroxidase-conjugated secondary antibody was then added to incubate the blots at room temperature for 2 h. At last, the blots were visualized with the Novex™ chemiluminescent substrate reagent kit (Waltham, MA, USA).

Cross-sections (7 μm cryosections from formalin-fixed samples) were washed in PBS (3 × 5 min), permeabilized in 0.5% Triton X-100 (30 min), and blocked for non-specific binding in 5% goat serum containing 1% BSA (30 min). The sections were then incubated with primary antihuman polyclonal antibodies against Ki67 (rabbit IgG, 1:200, Thermoscientific, RB-1510-P0), CD45 (mouse IgG1, 1:1000, Abcam, ab33533), vimentin (mouse IgM, 1:2000, Abcam, ab20346), αSMA (rabbit IgG, 1:600, Abcam, ab5694), or collagen I (mouse IgG1, 1:200, Sigma, c2456), in 10× diluted block solution (overnight at 4 °C). After washing in PBS (3 × 5 min), the sections were incubated with 1:500 secondary goat antibodies labeled with Alexa-488 conjugate (antimouse IgG1 (for CD45, Molecular Probes, A21121) or antirabbit IgG (for αSMA, Molecular Probes, A11008)) or Alexa-647 conjugate (antimouse IgM (for vimentin, Jackson Immunoresearch, 115-605-075) or antimouse IgG1 (for collagen I and Ki67, Molecular Probes, A21240)), in 10× diluted block solution (60 min). Nuclei were stained with 4′,6-diamidino-2-phenylindole (1:500, Sigma). The stained sections were mounted in mowiol (Sigma, 81381) and visualized with an inverted epifluorescent microscope (Zeiss Axiovert 200M, ×20/0.5 Plan-Neofluar lens, three sections/sample).

Cell proteins were extracted with RIPA lysis buffer (Thermo Fisher, USA). The protein was extracted by incubation, vortex, and centrifugation (15,000 ×g, 4°C for 25 min). A BCA reagent kit (Beyotime, China) was used to measure the protein concentrations. Total protein was separated by SDS-PAGE gel (100 V, 1.5 h) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA) (50 V, 80 min). After blocking in 5% nonfat milk for 1 hour, the membranes were incubated overnight at 4°C with the indicated primary antibodies, including anti-CD63 (Abcam, ab68418), anti-TSG101 (Abcam, ab133586), anti-E-cadherin (Abcam, ab76055), anti-N-cadherin (Abcam, ab76011), anti-vimentin (Abcam, ab20346), anti-JAK1 (Abcam, ab138005), anti-STAT3 (Abcam, ab68153), IL-10 (Abcam, ab215975), arginase-1 (ab133543) and anti-GAPDH (Abcam, ab9485). They were then incubated with secondary antibodies for 1 hour at room temperature and visualized by the ECL chemiluminescence reagent (Millipore, USA).

Lysis buffer was used to resolve the transfected cells, which were then centrifuged for 15 minutes at 4°C, 12000 rpm. A total of 50 μg proteins were added to 8%–15% polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5%–10% nonfat milk at room temperature for two hours with gentle agitation, the membranes were incubated with primary antibodies: anti‐GAPDH (1:1000, CST), anti‐LASP1 (1:500, ab1301, abcam), anti‐TGF‐β (1:500, ab92486, ab cam), anti‐E‐cadherin (1:1000, ab133597, abcam), anti‐N‐cadherin (1:1000, ab98952, abcam), anti‐Vimentin (1:500, ab20346, abcam), anti‐p‐Smad3 (1:2000, ab52903, abcam), and anti‐Smad3 antibody (1:1000, ab40854, abcam) at 4°C overnight. Subsequently, the corresponding secondary antibodies (1:2000, sc2030, Santa Cruz) were added to incubate the membranes for two hours at room temperature. They were then washed three times, and an enhanced chemiluminescence kit was used to detect the blots. GAPDH staining was used for normalization.

Cell proteins lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose (NC) membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature. Specific primary antibodies against E-cadherin (ab231303, 1:1,000, abcam) and Vimentin(ab20346, 1: 1,000, abcam)were added for overnight incubation at 4°C, followed by incubation with HRP-labeled secondary antibodies and Enhanced Chemiluminescence (ECL) Kit (Thermo Scientific, CA, USA) was used to measure the immunoreactivity.

Serial 4-μm-thick paraffin sections from CIA synovial tissues were deparaffinized in xylene and rehydrated through a graded ethanol series. Then, the sections were immersed in the 0.01 M citrate buffer (pH 6.0), and microwave irradiation was performed three times (8 min/time) for antigen retrieval. The sections were incubated in 5% goat serum for 1 h before immunostaining. In double-labeling experiments, the sections were incubated with anti-CD68 (pAb, 1:200, ab125212, Abcam, British; mAb, 1:100, NBP2-32831, NOVUS, USA), anti-Vimentin mAb (1:200, ab92547, Abcam, British; 1 : 200, ab20346, Abcam, British), anti-TLR2 pAb (1:150, 17236-1-AP, Proteintech, China), and anti-TLR4 mAb (1 : 100, sc-293072, Santa Cruz, USA) at 4°C overnight, followed by incubated with secondary antibodies, goat anti-rabbit IgG H&L(FITC) (1 : 200, ab6717, Abcam, British) or goat anti-mouse IgG H&L (Cy3) (1:200, ab97035, Abcam, British). The poststaining sections were examined with a full-spectrum scanning confocal microscope (FV1200, Olympus, Japan), and pairs of images were superimposed for colocalization analysis.