Ab231227

Heart tissue or cells were lysed with radioimmunoprecipitation (RIPA) lysis buffer. The prepared protein sample was separated through 10% SDS‐PAGE and then transferred to PVDF membrane (Millipore). 5% fat‐free milk was used for 1‐hour blocking at room temperature. The membrane was subsequently incubated with primary antibodies overnight at 4°C. The following day secondary antibodies were used for 1‐hour incubation at room temperature before the enhanced chemiluminescence (Millipore) with Amersham Imager 680 (General Electric Company). Primary antibodies included those against CD31 (Abcam, ab28364; Novus, NB100‐2284), VE‐cadherin (Abcam, ab231227; ab33168), α‐SMA (Abcam, ab5694), Vimentin (CST, #5741), collagen I (Abcam, ab34710), collagen III (Abcam, ab7778), Phospho‐AMPKα (Thr172) (CST, #2535), AMPKα (CST, #5831), Smad4 (CST, #38454), TGF‐β (Santa Cruz, sc130348), NOX4 (Proteintech, 14347‐1‐AP) and GAPDH (Proteintech, 10494‐1‐AP).

Lung tissue specimens were embedded in paraffin and sliced 2–4 μm thick. The sections were dewaxed, rehydrated, immersed in 0.01 M citrate buffer (pH 6.0), and boiled in a pressure cooker for 2 min. The sections were blocked with 3% hydrogen peroxide for 10 min and then incubated overnight at 4°C with rabbit monoclonal antibody against ZO-1 (1:400; catalog no. ab221547, Abcam, UK) or rabbit polyclonal antibody against VE-cadherin (1:100; catalog no. ab231227, Abcam). The sections were exposed to bio-goat antirabbit IgG as a secondary antibody from an SP (mouse/rabbit IgG)-POD kit (Catalog no. SP0041, Solarbio Life Sciences) following the manufacturer's instructions. Then, the sections were stained with DAB from the SP (mouse/rabbit IgG)-POD kit. Normal rabbit IgG (1:100, catalog no. AB-105-C, R&D) in PBS was used instead of a primary antibody (ZO-1 or VE-cadherin) in the immunohistochemistry procedure as a negative control. Spleen and kidney tissues instead of lung tissue were used for ZO-1 and VE-cadherin IHC as a positive control.
Expression of ZO-1 and VE-cadherin was visualized by a light microscope (EVOS FL AutoLife Technologies) and quantified from six randomly selected fields of view using Image-Pro-Plus 6. The integrated optical density (IOD) was measured from areas of protein staining, and the ratio of IOD area was used in statistical analysis.

The total protein in the lung tissue was extracted with cell lysis buffer and measured using a BCA protein assay kit, and the proteins were resolved using SDS–polyacrylamide gel electrophoresis. Electrophoresis of proteins was followed by transfer to PVDF membrane. The membrane was incubated with primary antibodies against Bax (ab32503, Abcam, China; 1 : 1000), Bcl-2 (ab32124, Abcam, China; 1 : 1000), cleaved caspase-3 (ab32042, Abcam, China; 1 : 1000), SDC-1 (ab128936, Abcam, China; 1 : 1000), claudin-5 (ab131259, Abcam, China; 1 : 1000), ZO-1 (ab190085, Abcam, China; 1 : 1000), and VE-cadherin (ab231227, Abcam, China; 1 : 1000) at 4°C overnight, followed by 1 hour at room temperature with the secondary antibody. The protein bands were identified using an enhanced detection chemiluminescence (ECL) technique.

Protein was isolated from the right lower lobe of lung tissues using a RIPA buffer (Beyotime, China) obeying the protocol of the manufacturer. Protein was then separated on 10% SDS-PAGE, transferred onto PVDF members (Millipore, USA), blocked with 5% nonfat milk, and probed with primary antibodies, such as PLCε-1 (ab109501, Abcam, USA), TLR4 (#14358, Cell Signaling Technology, USA), RelA (p65, #3039, Cell Signaling Technology, USA), caspase 3 (#9662, Cell Signaling Technology, USA), Bcl-2 (ab194583, Abcam, USA), Bax (ab32503, Abcam, USA), MLCK (ab76092, Abcam, USA), VE-cadherin (ab231227, Abcam, USA), occludin (ab216327, Abcam, USA), ZO-1 (ab191143, Abcam, USA), and GAPDH (ab8245, Abcam, USA) at 4°C overnight, respectively. Subsequently, the members were incubated with the secondary antibody goat anti-rabbit IgG HRP (ab6721, Abcam, USA) at room temperature for 1 h. Finally, the bands were visualized using an enhanced chemiluminescent agent (Bio-Rad, USA). The levels of protein were normalized using GAPDH.

The lung tissue sections were routinely dewaxed and hydrated; Claudin-5 antibody (1:200; ab131259; Abcam), ZO-1 antibody (1:300; ab96587; Abcam), and VE cadherin antibodies (1:300; ab231227; Abcam) were incubated overnight at 4°C and negative controls were incubated with PBS. Sections were covered with 3,3'-diaminobenzidine (DAB) tetrahydroxychloride and counterstained with hematoxylin. Under light microscopy, the tissue was dark brown for protein positive expression, and no staining or staining was negative or weakly positive. The average optical density (AOD) of the positive expression was analyzed by IPP software = integrated optical density (IOD)/positive expression area.