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The hypoxia, negative control, and Len control groups were kept in a hypoxic chamber (5% [O.sub.2], 5% C[O.sub.2], and 90% [N.sub.2]) for 24 h, then the total content of occludin and ZO-1 and the permeability of rGENCs were assessed.
The specific primers were the following: zo-1 (forward primer (F), 5'-ACCAGTAAGTCGTCCTGATCC-3' and reverse primer (R), 5'-TCGGCCAA ATCTTCTCACTCC-3'); claudin-1 (F, 5'-CGAGAGCTACAC GTTCACGG-3' and R, 5'-GGGT GTCGAGG GAAAAA TAGG-3'); cadherin-E (F, 5' -AAAGG CCCATTTCCTAAAAACCT-3' and R, 5'-TGCG TTCTCTATCCAGAGGCT-3'); cadherin-N (F, 5'-AGCC AACCTTAAC TGAGGAGT-3' and R, 5'-GGCAAGTT GATTGGAG GGATG-3'); and vimentin (F, 5'-GACGCC ATCAACACCGAGTT-3' and R, 5'-CTTTGTCGTTGGTT AG CTGGT-3').
After blocking with 5% BSA for 30 min, the membranes were incubated overnight at 4[degrees]C with the appropriate primary antibodies for [beta]-actin (Santa Cruz, CA), MLCK, phosphorylated MLCK, ZO-1, occludin, and F[beta]-actin (Cell Signaling Technology), followed by incubation with a horseradish peroxidase- (HRP-) conjugated secondary antibody for 1 h.
A BCA protein quantification kit, fluorescence-tagged antibody and anti-zonula occludens-1 (ZO-1), anti-occludin antibody, and anti-F4/80 antibody were purchased from Thermo Fisher Scientific and Santa Cruz Biotechnology (Dallas, TX, USA).
ZO-1 belongs to the membrane-associated guanylate kinase family of proteins and usually acts as a scaffold and recruits various signaling molecules and the actin cytoskeleton to TJs.[sup] Rhubarb, a traditional Chinese herb, has been used to treat diarrhea in traditional Chinese medicine,[sup] but the pharmacological mechanism is unclear.
Immunofluorescence and Quantification of ZO-1 Deposits.
Antibodies against ZO-1, ERK1/2, and phosphorylated ERK1/2 were purchased from Cell Signaling Technology (CST) (Danvers, USA).
Antibodies (anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), occludin, claudin-1, ZO-1, phosphorylated Akt [p-Akt], and phosphorylated mTOR [p-mTOR]) were from Santa Cruz.
ZO-1 staining was observed in the tight junction complexes of adjacent PECs and foot processes of podocytes (Figure 3(b)).
Chromogenic in Situ Hybridization (CISH) was done to detect Occludin and ZO-1 mRNA The protein weight was measured by comparing protein fraction in Fermentes standard.
At different time points, the expression of claudin-5, occludin, ZO-1, and PKCa signaling pathway in microvessel fragments of cerebral ischemic tissue were evaluated.
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