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).
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
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.
(12) Hay muy pocos estudios de la variacion del gen TJP1 que codifica para ZO-1
; se han reportado cuatro polimorfismos en regiones intronicas asociados con los desordenes de espectro autista, queratocono y respuesta a los antipsicoticos.
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.