Anti occludin 13409 1 ap

HUVECs were washed with PBS and lysed in RIPA buffer (20 mMTris pH 7.5, 150 mM NaCl, 50 mM NaF, 1% NP40, 0.1% DOC, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, 1 μg/mL leupeptin). Protein concentration was determined using the BCA assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by 10% SDS-PAGE gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. PVDF membranes were blocked with nonfat dry milk in PBS containing 0.05% Tween-20 and incubated overnight with a primary antibody in PBS-T at 4 °C. Primary antibodies were rabbit anti-human vWF antibody (A008229; 1:800; Dako, Glostrup, Denmark), anti-occludin (13409–1-AP; 1:3000; Proteintech, Wuhan, China), and anti-β-actin (20536–1-AP; 1:8000; Proteintech). Immunoreactivity was visualized with HRP-conjugated secondary antibodies and chemiluminescence (ECL kit, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

The protein expressions of Occludin and β defensin in the colon wall were determined by the standard Western blot method with the GAPDH protein as a loading control. The anti-Occludin (#13409-1-AP) and anti-GAPDH (10494-1-AP) primary antibodies were obtained from Proteintech (Wuhan, Hubei, China). The anti-β defensin (#bs-1296R) primary antibody was obtained from Bioss (Bioss Biotech, Beijing, China). The horseradish peroxidase-conjugated secondary antibody was obtained from Cell Signaling Technology Co.

For Western blot or immunofluorescence analysis, the following antibodies were used: anti-ADAM10 (A10438; ABclonal, Wuhan, China), anti-E-cadherin full length (3195; Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin NTF (A3044, ABclonal), anti-claudin-1 (ab129119; Abcam, Cambridge, MA, USA), anti-occludin (13,409-1-AP; Proteintech, Wuhan, China), anti-ZO-1 (21,773-1-AP; Proteintech), anti-HtrA1 (ab199529; Abcam), anti-MMP7 (10,374-2-AP; Proteintech), anti-FLAG (20,543-1-AP; Proteintech), and anti-GAPDH (10,494-1-AP; Proteintech). Horseradish peroxidase (HRP)-labeled goat anti-rabbit (bs-0295 G-HRP; Bioss, Beijing, China), HRP-labeled goat anti-mouse (bs-0296 G-HRP; Bioss), and Cy3-conjugated goat anti-rabbit (A0516; Beyotime) secondary antibodies were used.