Journal: International journal of molecular sciences

Article Title: Restraint Stress Disrupted Intestinal Homeostasis via 5-HT/HTR7/Wnt/β-Catenin/NF-kB Signaling.

doi: 10.3390/ijms26094021

Figure Lengend Snippet: Figure 4. 5-HT activated Wnt/β-catenin by acting on the HTR7 receptor. The expression of β-catenin and β-actin protein were examined in jejunum (a) and colon (b) by Western blot, and relative protein levels were normalized to β-actin (n = 3). Immunofluorescence staining of β-catenin in Caco2 cells (c) and total and nuclear β-catenin intensities were analyzed by ImageJ (d) (n = 6 hole). 5-HT and SB269970 treated Caco2 cells were used for β-catenin coimmunoprecipitation, input samples from the coimmunoprecipitation assay were analyzed by Western blot (e), and relative protein levels were normalized to β-actin (f) (n = 3). The expressions of TPH1, APC, Axin1, GSK3-β, β-catenin and β-actin protein were examined in jejunum (g) by Western blot, and relative protein levels were normalized to β-actin (h) (n = 3). The expression of β-catenin, TCF3, APC, Axin1, GSK3-β, TPH1, SERT, HTR7 and β-actin protein were examined in colon (i,l) by Western blot, and relative protein levels were normalized to β-actin (j,k,m) (n = 3). Each sample was assayed three times. Data are presented as the mean ± SEM. Differences were assessed by ANOVA and denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001 indicate significant difference. C: control group; S: restraint stress; S + PCPA: restraint stress + PCPA; S + IWP-2: restraint stress + IWP-2.

Article Snippet: The membrane was then blocked with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 1.5 h. Subsequently, the membrane was incubated overnight at 4 ◦C with the antibodies: rabbit anti-Claudin-3 antibody (1:1000, ab15102, Abcam, Cambridge, CA, USA), rabbit antiClaudin-1 antibody (1:3000, ab15098, Abcam, Cambridge, CA, USA), rabbit anti-Occludin antibody (1:1000, ab216327, Abcam, Cambridge, CA, USA), rabbit anti-TPH1 antibody (1:1000, ab52954, Abcam, Cambridge, CA, USA), rabbit anti-SERT antibody (1:1000, 19559- 1-AP, Proteintech, Wuhan, China), rabbit anti-HTR7 antibody (1:1000, DF13323, Affinity, OH, USA), mouse anti-β-catenin (1:8000, 66379-1-AP, Proteintech, Wuhan, China), rabbit anti-APC antibody (1:1000, ab40778, Abcam, Cambridge, CA, USA), rabbit anti-Axin1 antibody (1:1000, C76H11, Cell Signaling Technology, Beverly, MA, USA), rabbit anti-GSK3β antibody (1:1000, SC-377213, Santa Cruz, Dallas, TX, USA), rabbit anti-TCF3 antibody (1:2000, 21242-1-AP, Proteintech, Wuhan, China), rabbit anti-Phospho-IkB (1:1000, ab133462, Abcam, Cambridge, CA, USA), rabbit anti-Phospho-NF-kB p65 (1:1000, ab76302, Abcam, Cambridge, CA, USA), mouse anti-β-actin antibody (1:10,000, 66009-1-lg, Proteintech, Wuhan, China), rabbit anti-Lgr5 (1:1000, DF2816, Affinity Biosciences, Jiangsu, China), rabbit anti-Axin2 (1:1000, 20540-1-AP, Proteintech, Wuhan, China), and mouse anti-c-Myc (1:1000, sc-40, Santa Cruz, Dallas, TX, USA) overnight at 4 ◦C.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Control