Journal: PLoS ONE
Article Title: Antioxidant activity and laxative effects of tannin-enriched extract of Ecklonia cava in loperamide-induced constipation of SD rats
Figure Lengend Snippet: Expressions of mAChRs and key mediators within their downstream signaling pathway in Lop+TEE treated constipation rats. Expression levels of mAChRs and key mediators, including mAChR M2, mAChR M3, Gα, PKC, p-PKC, PI3K and p-PI3K, in the mAChR M2 and M3 signaling pathway were measured by Western blot analysis using specific primary antibodies and HRP-labeled anti-rabbit IgG antibody. After the intensity of each band was determined using an imaging densitometer, relative levels of the seven proteins were calculated based on the intensity of actin. Four to six rats per group were used in the preparation of the total tissue homogenate, and Western blot analyses were assayed in duplicate in each sample. The data are reported as the mean ± SD. *, p
Article Snippet: Proteins (30 μg) were subjected to 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, and the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. The membranes were then probed with the following primary antibodies, overnight at 4°C: anti-Gα (ab128900, 1:1,000, Abcam, Cambridge, UK), anti-mAChR M2 (AMR-002, 1:1,000, Alomone Labs, Jerusalem, Israel), anti-mAChR M3 (AMR-006, 1:1,000, Alomone Labs), anti-PKC (2058s, 1:1,000, Cell Signaling Technology Inc., Danvers, MA, USA), anti-p-PKC (9376s, 1:1,000, Cell Signaling Technology Inc.), anti-PI-3K (4292s, 1:1,000, Cell Signaling Technology Inc.), anti-p-PI3K (4228s, 1:1,000, Cell Signaling Technology Inc.), anti-SOD (ab13498, 1:1,000, Abcam), anti-Nrf2 (ab137550, 1:1,000, Abcam), anti-p-Nrf2 (PA5-67520, 1:1,000, Invitrogen Co., Carlsvad, CA, USA), or anti-actin (4967s, 1:3,000, Sigma-Aldrich Co.).
Techniques: Expressing, Western Blot, Labeling, Imaging