Mouse monoclonal anti occludin

10x10⁴ cells were seeded on glass coverslips precoated with collagen in 24-well plates, allowed to attach overnight, and treated with SH extract according to protocol. For detection of β-catenin and occludin proteins, cells were exposed to UVB radiation, as described in the protocol above. After irradiation, cells were fed with fresh growth medium or used for experiment following the protocol. For immunofluorescence assay, 4 hours after the UVB irradiation, cells were washed, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS and stained for 1 hour at 37° C with different protein-specific antibody: mouse monoclonal anti-occludin Invitrogen Life Technologies, Waltham, MA, USA mouse anti-filaggrin (Sigma-Aldrich, St Louis, MO, USA). After PBS wash, cells were incubated with secondary antibodies/fluorescein isothiocyanate (Alexa Fluor 488 anti-mouse or Alexa Fluor 536 anti-rabbit immunoglobulin G, Molecular Probes, Invitrogen Life Technologies, Waltham, MA, USA) and with phalloidin for 1 hour at room temperature. Following 10 minutes of treatment with RNase, the coverslips were mounted on glass slides by using Mowiol 40–88 (Sigma-Aldrich, St Louis, MO, USA) added of propidium iodide. Images were acquired through confocal microscope LSM 800, magnification 60X, software ZN 2.1 blue Edition (Carl Zeiss, Jenza, Germany) and quantified using ImageJ software.

Mouse monoclonal antihuman JAM‐A was purchased from NovusBio (Littleton, CO; #H00050848‐M01). Goat polyclonal antihuman JAM‐A (#AF1033) and goat polyclonal antimouse JAM‐A (#AF1077) were both purchased from R&D Systems (Minneapolis, MN). Mouse monoclonal antioccludin, mouse monoclonal antizonula occludens‐2 (ZO‐2), and donkey polyclonal antigoat IgG (H+L)‐Alexa Fluor® 488 were purchased from Thermo Fisher (#33‐1500, #37‐4700, and #A‐11055). Mouse monoclonal anti‐β‐actin and mouse monoclonal antimyosin light chain kinase (MLCK) were purchased from Sigma‐Aldrich (#A5441 and #M7905). Mouse monoclonal anti‐FLAG (Sigma‐Aldrich #F1804) was a kind gift from Dr. Chris Yun (Emory University, Atlanta, GA). Goat antimouse IgG‐HRP was purchased from Santa Cruz (Dallas, TX; #sc‐2005).

Rabbit polyclonal anti-ZO-1, mouse monoclonal anti-occludin, and mouse monoclonal anti-claudin5 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit monoclonal anti-β-actin, anti-phospho-NF-κB p65, and anti-NF-κB p65 were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat polyclonal anti-Axl, anti-Mertk, and rat monoclonal anti-Tyro3 antibodies were obtained from R&D Systems (Minneapolis, MN, USA). Rabbit polyclonal anti-phospho-Axl, anti-phospho-Mertk, and anti-phospho-Tyro3 were purchased from Affinity (Cincinnati, OH, USA). Horseradish peroxidase (HRP)-conjugated and Alexa Fluor-conjugated secondary antibodies were purchased from Biosharp (Hefei, China). Axl small interfering RNA (siRNA) or nonspecific control siRNA with the recommended transfection reagents were all from RiboBio (Guangzhou, China). LPS (O55:B5) was purchased from Sigma-Aldrich (Oakville, Ontario, Canada). The In Vitro Vascular Permeability Assay kit was obtained from Merck Millipore (Etobicoke, Ontario, Canada). Recombinant Mouse Gas6 protein was from R&D Systems (Minneapolis, MN, USA). The chemiluminescence (ECL) Western blot detection kit was from Wenke Biotechnology (Zhejiang, China).

Mouse monoclonal anti-occludin (#33-1500) and rabbit polyclonal anti-ZO-1 (#40-2200) antibodies were purchased from ThermoFisher Scientific (Carlsbad, CA, USA). Mouse monoclonal anti-E-cadherin (#610404) and rabbit polyclonal anti-β-catenin (#610153) antibodies were purchased from BD Biosciences (San Jose, CA, USA). HRP-conjugated anti-mouse IgG (#12-349), HRP-conjugated anti-rabbit IgG (#12-348), anti-β-actin (#A5541) antibodies, AlexaFlour-488-conjugated anti-mouse IgG (#62197), Cy3-conjugated anti-rabbit IgG (#AP132C), rabbit polyclonal anti-JNK2(pT183/pY185) (SAB4300198) antibodies, and AlexaFlour-488-conjugated phalloidin (#49409) were purchased from Millipore Sigma (Eugene, OR, USA). All antibodies were affinity purified using the immunogen ligands. The specificity of most antibodies was tested by the siRNA-mediated knockdown of target proteins in the cells. The antibodies used have been extensively used in our laboratory as well as in other laboratories. The non-specific binding of fluorescently labeled secondary antibodies was tested by incubating cell monolayers with secondary antibodies without the primary antibodies.