Journal: Shock (Augusta, Ga.)
Article Title: INTESTINAL EPITHELIAL-SPECIFIC OCCLUDIN DELETION WORSENS GUT PERMEABILITY AND SURVIVAL FOLLOWING SEPSIS
doi: 10.1097/SHK.0000000000002531
Figure Lengend Snippet: Levels of occludin (A) were markedly decreased in occludin KO IEC mice ( P = 0.002). ZO-1 expression was significantly increased in occludin KO IEC mice following sepsis (B, P = 0.002) despite there being no difference between knockout and WT mice at baseline (C, P = 0.49). In contrast, protein levels of tricellulin (D, P = 0.82) and JAM-A (E, P > 0.99), were similar between occludin KO IEC and WT mice following sepsis as was the ratio of pMLC/MLC (F, P = 0.59). Representative Western blot images are shown for each protein with β-actin levels shown as loading control, n = 6/group.
Article Snippet: Membranes were blocked with 3% (w/v) bovine serum albumin or 3% (w/v) nonfat dry milk in TBS buffer for 1 h at room temperature, followed by incubation at 4°C overnight with the following primary antibodies: rabbit polyclonal anti-occludin (1:500, 71–1500, Thermo Fisher Scientific), rabbit polyclonal anti-ZO-1 (1:500, 61–7300, Thermo Fisher Scientific), rabbit polyclonal anti-tricellulin (1:500, 48–8400, Thermo Fisher Scientific), rabbit polyclonal anti-JAM-A (1:1,000, 36–1700, Thermo Fisher Scientific), mouse monoclonal anti-phospho-myosin light chain (pMLC) (1:1,000, 3675, Cell Signaling Technology), rabbit anti-myosin light chain (MLC) (1:1,000, 3672, Cell Signaling Technology), mouse monoclonal anti-claudin 2 (1:1,000, 32–5600, Thermo Fisher Scientific), rabbit polyclonal anti-claudin 4 (1:500, 36–4800, Thermo Fisher Scientific), rabbit polyclonal anti-claudin 5 (1:2,000, 34–1600, Thermo Fisher Scientific), rabbit polyclonal anti-claudin 8 (1:2,000, 40–0700Z, Thermo Fisher Scientific), rabbit polyclonal anti-claudin 12 (1:500, 38–8200, Thermo Fisher Scientific), rabbit polyclonal anti-claudin 15 (1:500, 38–9200, Thermo Fisher Scientific), rabbit polyclonal anti-Bax (1:1,000, 2772, Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-Bcl-2 (1:1,000, 2876, Cell Signaling Technology), rabbit polyclonal anti-cleaved capase-3 (1:1,000, 9661, Cell Signaling Technology), and rabbit polyclonal anti-β-actin (1:1,000, 4967, Cell Signaling Technology).
Techniques: Expressing, Knock-Out, Western Blot, Control
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