Western Blot Analysis of Tight Junction Proteins

Cells were lysed as previously described^{62 (link),63 (link)}. Proteins were quantified with the Bradford assay. Proteins were separated onto a pre-cast 4–20% polyacrylamide gel (Mini-PROTEAN^® TGX gel, Bio-rad) and transferred to PVDF membranes (Trans-Blot^® TurboTM PVDF Transfer packs, Bio-rad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (1:5000; ab8227, Abcam), ZO-1 (1:300; ab96587, Abcam), occludin (1:5000; ab31721, Abcam) and TLR-2 (1:500; Santa Cruz, Dallas, TX, USA) were used. Secondary antibodies were obtained from Abcam (1:5000; anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with ECL reagents (Clarity^TM Western ECL Blotting Substrate, Bio-rad). Densitometry was performed by ImageJ software. All blots derive from the same experiment and they were processed in parallel.