Anti claudin 1 antibody

Cell infections were performed after 36 h of 5µM of SC-236 treatment as described earlier. After infection, cells were fixed in 4% PFA for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS. Thereafter, cells were blocked for an additional 60 min with the 5% BSA. Cells were stained with anti-psoriasin antibody (Santa Cruz Biotechnology), anti-hBD2 antibody (Santa Cruz Biotechnology), anti-KEAP1 antibody (Proteintech), anti-claudin-1 antibody (Invitrogen), anti-CD86 antibody (BD Biosciences) and anti-TLR4 (BD biosciences) at 1:200 dilutions or anti-NRF2 (Cell Signaling Technologies) at 1:100, or with anti-NOS2 antibody (Santa Cruz Biotechnology) at 1:50 dilution followed by the respective Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 647 conjugated secondary antibody (Life Technologies) at 1:400 dilution and counter-stained using 2.5 µg mL^− 1 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Confocal images were acquired on a LSM 700 microscope (Carl Zeiss) using 63× oil immersion objective. All images were processed for intensity quantification by ImageJ software (NIH).

Excised ileal section was fixed in 10% neutral buffer formalin for 24 hours and embedded in paraffin. 8 µm section was mounted on microscopy glass slide and stained with hematoxylin and eosin (H&E) for histological evaluation. H&E-stained slides were randomized and coded for histological evaluation in a blinded fashion. For immunofluorescence staining with tight junction protein, fixed ileal sections were deparaffinized using xylene and passed through series of graded alcohol for rehydration. Heat antigen retrieval was performed using citrate antigen retrieval buffer (DAKO) and tissue section was blocked with 5% BSA. Ileal tissue sections were stained with anti-Claudin-1 antibody (Invitrogen, catalog no. 51–9000) at a dilution of 1:100 in PBS + 1%BSA overnight at 4°C. Anti-rabbit Alexa 488 conjugated secondary antibody (Invitrogen, catalog no. A-11008) was added at a dilution of 1:800 for 1 hour at room temperature. Ileal tissue section was mounted under coverslip using ProLong Gold antifade mounting media with DAPI (Invitrogen, catalog no. P36935) and visualized using fluorescence microscope (Leica Microsystems, Germany).

The intestinal samples were mixed with phosphate saline buffer (pH 7.4) containing 1 mmol/l PMSF and 1 mmol/l EDTA-free complete protease inhibitor and then homogenized and centrifuged at 4°C 12,000 rpm for 20 min. The supernatant fluid was collected and electrophoresed on sodium dodecyl sulfate–polyacrylamide gels, transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore, Billerica, MA, USA), and then probed with the anti-E-cadherin antibody (Abcam, Cambridge, MA, USA) and anti-claudin-1 antibody (Invitrogen, Carlsbad, CA, USA). Signals were detected with an enhanced chemiluminescence system (Millipore, Billerica, MA, USA).