Secondary peroxidase linked goat anti rabbit igg

Total proteins extracts of each group cells were resolved by 10 % SDS-PAGE and transferred on PVDF (Millipore) membranes. After blocking, the PVDF membranes were washed 4 times for 15 min with TBST at room temperature and incubated with primary antibody (rabbit anti-Id-1 polyclonal antibody Abcam). Following extensive washing, membranes were incubated with secondary peroxidase-linked goat anti-rabbit IgG (Santa Cruz) for 1 h. After washing 4 times for 15 min with TBST at room temperature once more, the immuno-reactivity was visualized by enhanced chemiluminescence (ECL kit, Pierce Biotechnology), and membranes were exposed to KodakXAR-5 films (Sigma-Aldrich).

Ear tissues of mice were removed and ground into homogenates with 20 μl/mg protein lysis solution (RIPA, phenylmethylsulfonyl fluoride and phosphatase inhibitor; 100:1:1). As for in vitro experiment, HaCaT cells were scraped from six‐well plates containing 100 μl of RIPA: phenylmethylsulfonyl fluoride (100:1). The samples (either from ear tissues or cells) were collected in microcentrifuge tubes, and lysed for 20 min. Then the homogenates were centrifuged at 13,201 g at 4°C for 10 min. The protein concentrations of the tissue or cell samples were determined using a BCA protein assay kit (Thermo scientific). Total protein extracts were resolved by 20% SDS–PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). After blocking with skim milk, the membranes were washed five times for 5 min. with Tris‐buffered saline, containing 0.1% Tween‐20 (TBST) at room temperature and then incubated with antibodies against CLDN‐1, CLDND1 or occludin (1:1000 dilution; Abcam) at 4°C overnight. After washing, membranes were incubated at room temperature with secondary peroxidase‐linked goat anti‐rabbit IgG (1:1000 dilution; Santa Cruz Biotechnology) for 2 hr. After washing, protein bands were detected by enhanced chemiluminescence (ECL kit; Millipore) and the protein expressions were quantified by ChemiScope analysis.

Total proteins extracts of each group cells were resolved by 12% SDS-PAGE and transferred on PVDF (Millipore) membranes. After blocking, the PVDF membranes were washed 4 times for 15min with TBST at room temperature and incubated with primary antibody. Following extensive washing, membranes were incubated with secondary peroxidase-linked goat anti-rabbit IgG (1:1000, Santa Cruz) for 1h. After washing 4 times for 15min with TBST at room temperature once more, the immunoreactivity was visualized by enhanced chemiluminescence (ECL kit, Pierce Biotechnology), and membranes were exposed to Kodak XAR-5 films (Sigma-Aldrich Chemical).