Anti α sma antibody

RIPA lysis buffer (strong) RIPA (YEASEN, 20101ES60, USA) was used to extract proteins from mechanically stretched and unstretched AD‐VSMCs. The protein concentration was determined using the BCA method, and Western blotting was then used to detect protein levels. Briefly, protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were then blocked with 5% skim milk for 2 h and incubated with primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with secondary antibodies for 1 h. Visualization was performed using an enhanced chemiluminescence detection system, and Photoshopcs3 software was used to analyze the relative band intensity. The primary antibodies used in this study were as follows: anti‐α‐SMA antibodies (Abcam, Cambridge, UK; cat. no. ab7817), anti‐SM22‐α antibodies (Abcam; cat. no. ab14106), anti‐OPN antibodies (Abcam; cat. no. ab8448), anti‐PCNA antibodies (Abcam; cat. no. ab29), anti‐KLF4 (Abcam; cat. no. 106629), anti‐MMP9 antibody (Abcam; cat. no. ab76003), anti‐GAPDH antibody (Abcam; cat. no. ab9485) and anti‐β‐actin antibody (Arigo; cat. no. arg62346).

Preparation of liver tissue sections was performed as described previously [44 (link)]. The sliced liver paraffin sections were then stained with H&E and Sirius Red. For immunohistochemistry (IHC), cryostat sections were incubated with anti-F4/80, anti-CD11b and anti-Gr-1 antibodies (BD Pharmingen, San Diego, CA, USA) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. For fluorescence staining, cryostat sections were incubated with anti-collagen I, anti-MyD88 and anti-α-SMA antibodies (Abcam, Cambridge, Cambs, UK) followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Sections were evaluated under the bright-field and fluorescent microscope (DP71, Olympus, TKY, JPN).
Liver tissues of cirrhosis patient cases for Sirius red and MyD88 staining were from the First Affiliated Hospital of China Medical University (Shenyang, Liaoning, CN). The hospital provided ethical statements to confirm that the local ethics committees approved their consent procedures, and all participating patients signed an approved informed consent form. The ethical statement provided by the hospital and the protocol of the experiment were checked carefully and approved by the Ethics Committee of Beijing Jiaotong University.

For the histopathological examination, after the extraction of the left lung, it was immediately fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 μm thick slices. The right lung was obtained to determine the wet-to-dry lung weight ratio. The lung tissues were stained with hematoxylin and eosin and Masson's trichrome according to the manufacturers' protocols. Immunostaining with anti-α-SMA antibodies (Abcam) was performed with the Novolink DAB Polymer Detection system (Leica Biosystems Newcastle Ltd) according to the manufacturer's recommendations. Endogenous peroxidase activity was neutralized using the Peroxidase Block reagent (hydrogen peroxide; Novolink DAB Polymer Detection system; Leica Biosystems Newcastle Ltd). Rabbit anti-mouse IgG was used as a secondary antibody (Novolink DAB Polymer Detection system).

LX-2 cells were lysed in RIPA buffer containing 1% PMSF and protease inhibitor cocktail. The cell lysate was centrifuged at 12000 rpm for 20 min at 4 degree and the supernatant was collected. Protein concentration was determined by using BCA protein assay. A total of 40 μg of cell lyse were separated by SDS-PAGE gel and transferred to NC membranes. The membranes were incubated with anti-CD44s and, anti-α-SMA antibodies (Abcam, Cambridge, MA, USA), anti-β-actin antibody (Santa Cruz, Dallas, TX, USA). After washing 3 times with TBS/T buffer, membranes were incubated with a horseradish peroxidase-conjugated secondary antibodies (Boster, Wuhan, China) for 1 h. The blots were developed with ECL (Pierce) according to the manufacturer’s instruction.

LX-2 cells were plated into 24-well plates. For fluorescence staining, the cells were fixed in 4% formaldehyde for 15 min and permeabilized with 0.2% Triton-X 100 for 10 min at room temperature. The cells were incubated with 2% BSA to block nonspecific binding sites. Then, the cells were incubated with anti-MyD88, anti-α-SMA antibodies (Abcam, Cambridge, Cambs, UK) and anti-CD206 antibodies (BD Pharmingen, San Diego, CA, USA), followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA).

Immunohistochemistry (IHC) was performed to examine proliferating cell nuclear antigen (PCNA) and α-SMA levels. After deparaffinizing the section, pepsin (Sigma, USA) was used for antigen retrieval at 37 °C for 30 min. Then, 3% H₂O₂ was applied to block endogenous peroxidase activity. Mouse monoclonal anti-PCNA antibodies and anti-α-SMA antibodies (Abcam, UK) were added and incubated at 4 °C overnight. Sections were then incubated with goat anti-mouse IgG-HRP (Invitrogen, USA) for 1 h at room temperature. Finally, sections were stained with a DAB detection kit (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin.

Proteins were extracted from cell lysates in lysis buffer and used to quantify protein levels. Proteins were separated on polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoprobed with specific antibodies. The proteins were detected using a chemiluminescence reagent kit purchased from Thermo Fisher Scientific. The imager station captured the images (Odyssey Software Version 5.2, LI-COR Biosciences). Anti-TRIO (Affinity, DF2685), anti-TRIOBP (Proteintech, 16124-1-AP), anti-β-actin (Affinity, AF7018), and anti-GAPDH (Affinity, AF7021) antibodies were purchased from Affinity Biosciences LTD. Anti-α-SMA antibodies were purchased from Abcam (Abcam, ab124964). Anti-vimentin (Proteintech, 10366-1-AP), anti-E-cadherin (Cell Signaling Technology, 14472S), anti-type I collagen (Proteintech, 14695-1-AP) and anti-fibronectin (Proteintech, 15613-1-AP) antibodies were purchased from Proteintech Group.