Anti cav1

Total proteins were extracted using RIPA lysis buffer (Pierce, Invitrogen, Gaithersburg, MD, USA). The concentration of the extracted protein was determined by a BCA assay. The total protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a PVDF membrane (EMD Millipore, Billerica, MA, USA), which was blocked with 5% skim milk for 1 h. The primary antibodies, anti-VCAM1 (1 : 1000 dilution, Cell Signaling Technology, MA, USA), anti-VASP (1 : 1000 dilution, Cell Signaling Technology), anti-PICK1 (1 : 1000 dilution, Cell Signaling Technology), anti-FLNA (1 : 1000 dilution, Cell Signaling Technology), anti-COL4A2 (1 : 1000 dilution, Cell Signaling Technology), anti-CAV1 (1 : 1000 dilution, Cell Signaling Technology), and anti-GAPDH (1 : 1000 dilution, Cell Signaling Technology), were added and incubated with the membranes at 4°C overnight. Then, the membranes were washed with PBS buffer and incubated with anti-rabbit IgG antibody (1 : 10,000 dilution, Cell Signaling Technology) at 37°C for 45 min. An imaging system (Odyssey, LI-COR Biosciences, Lincoln, NE, USA) was used for the semiquantitative analysis. GAPDH was used as an internal control.

All the sections were blocked with 10% donkey serum with 0.3% Triton X-100 and 1% BSA for 2 h and incubated with anti-Cav1 (1:400, Cell Signaling Technology, MA, USA), anti-p-p65 (1:200, ABclonal, MA, USA), anti-COX-2 (1:200, HuaBio, Zhejiang, China), anti-Collagen I (1:200, Abcam, Cambridge, UK) and anti-IgG (1:400, Cell Signaling Technology, MA, USA) antibodies at 4 °C overnight, followed by incubation with secondary antibody (HRP conjugated goat anti-Rabbit IgG, 1:500, Beyotime, Shanghai, China) for 2 h. Sections were visualized with DAB (Sangon Biotech, Shanghai, China) for 30 min and counterstained with hematoxylin. Evaluation of the immunocytochemistry was done by light microscopy.

For western blotting, anti-beta-actin (1:1,000; cat. no. D6A8; Cell Signaling Technology, Danvers, MA, USA), anti-CAV1 (1:1,000; cat. no. D46G3; Cell Signaling Technology), anti-SLO (1:1,000; Cosmo Bio), and anti-Nga (1:1,000; cat. no. 64-005; Bio Academia) antibodies were used as primary antibodies, and horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies (1:5,000; Jackson ImmunoResearch Laboratories) were used as secondary antibodies. For immunostaining experiments, anti-CAV1 (1:400; cat. no. D46G3; Cell Signaling Technology) and anti-GAS (1:250 or 1:500; cat. no. PAB13831; Abnova and cat. no. ab9191; Abcam) antibodies were used as primary antibodies, and anti-mouse, anti-rabbit, or anti-goat IgG conjugated with AlexaFluor-488 and AlexaFluor-568 (1:250; Molecular Probes/Invitrogen) or AlexaFluor-594 (1:250; Jackson ImmunoResearch Laboratories) antibodies were used as secondary antibodies. 4′,6-Diamidino-2-phenylindole (DAPI; 1:1,000; Dojindo) was used to stain bacterial and cellular DNA.

Immunohistochemistry staining of the paraffin sections was performed using the ChemMate EnVision system (Dako, Glostrup, Denmark). The sections were deparaffinized in xylene and rehydrated in ethanol. Antigens were retrieved by boiling the sections in 0.5 M EDTA (pH 8) using a pressure cooker for 10 min and under high pressure for 3 min. After cooling to RT, endogenous peroxidase was blocked by incubating the sections with 0.3% hydrogen peroxide for 10 min. The sections were incubated with the primary antibody anti-CAV1 (1:400; D46G3 rabbit, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. After washing with TBS, the sections were incubated with EnVision Plus (Dako, Glostrup, Denmark) at RT for 30 min. The signals were developed using 3, 3′-diaminobenzidine (Dako, Glostrup, Denmark), and the sections were counterstained using hematoxylin.