Fig 1: ALK5 interacts with Gadd45b after I/R; a schematic diagram depicting the possible mechanism of ALK5 signaling regulating neurogenesis after cerebral I/R.a Coimmunoprecipitation experiments of ALK5 and Gadd45b in the ischemic cortex 24 h after I/R. b Coimmunoprecipitation experiments of ALK5 and Gadd45b in the ischemic cortex 14 d after I/R. These results show that ALK5 coimmunoprecipitated with Gadd45b after cerebral I/R. c Ischemia activates ALK5, which catalyzes the phosphorylation of Smad2/3 and forms a complex with Smad4. In the nucleus, the complex promotes Gadd45b expression, which elevates the expression and demethylation of BDNF, thereby improving neurogenesis
Fig 2: Lentiviruses are successfully transfected into neurons and effectively alter the expression of ALK5; ALK5 improves neurological function recovery.a Fluorescent images of GFP (green) in injected cortex 14 d after lentiviruses were stereotaxically injected (scale bar = 100 µm). b Representative Western blot images of ALK5 expression with LV-ALK5-RNAi or LV-con-RNAi injection 24 h and 14 d after I/R (n = 6 biological replicates). c Comparison of mean intensity ratios in Western blot analysis for ALK5 knockdown. d Representative Western blot images of ALK5 expression with LV-ALK5 or LV-con-ALK5 injection 24 h and 14 d after I/R (n = 6 biological replicates). e Comparison of mean intensity ratios in Western blot analysis for ALK5 overexpression. *P < 0.05, compared to the LV-con-RNAi or LV-con-ALK5 group at the same time point (Student’s t test). f, g The mNSS test and adhesive-removal somatosensory test were performed 3 d before and 1, 7, and 14 d after I/R (n = 10 biological replicates). There were no significant differences among the groups 1 d after I/R. However, there were significant differences among the groups 7 d and 14 d after I/R. *P < 0.05, compared to the LV-con-RNAi group at the same time point; ?P < 0.05, compared to the LV-con-ALK5 group at the same time point; #P < 0.05, compared to the MCAO/R group at the same time point (ANOVA)
Fig 3: ALK5 is increased in the ischemic hemisphere in a MCAO/R rat model.a Representative images of ALK5 expression in the ischemic hemisphere 24 h after I/R (n = 6 biological replicates). b Representative images of ALK5 expression in the ischemic hemisphere at 14 d after I/R (n = 6 biological replicates). c Comparison of mean intensity ratios in Western blot analysis. d Representative images of immunohistochemical staining for ALK5 expression 24 h and 14 d after I/R (n = 5 biological replicates) (scale bar = 100 µm). e Comparison of the mean density value in immunohistochemical analysis for ALK5 expression. f Representative images of immunofluorescence staining for ALK5 (green), beta-III tubulin (red)/GFAP (red) and cellular nuclei (blue). (scale bar = 100 µm). Arrows show the positive cells, and the inserted images show magnified images of representative cells. *P < 0.05, compared to the sham group at the same time point (Student’s t test)
Fig 4: ALK5 regulates the phosphorylation of Smad2/3 and the protein levels of Gadd45b.a Representative Western blot images and bar graph summary of Smad2/3 phosphorylation with LV-ALK5-RNAi or LV-con-RNAi injection 24 h and 14 d after I/R (n = 6 biological replicates). b Representative Western blot images and bar graph summary of Smad2/3 phosphorylation with LV-ALK5 or LV-con-ALK5 injection 24 h and 14 d after I/R (n = 6 biological replicates). c Representative Western blot images and bar graph summary of Gadd45b expression with LV-ALK5-RNAi or LV-con-RNAi injection 24 h and 14 d after I/R (n = 6 biological replicates). d Representative Western blot images and bar graph summary of Gadd45b expression with LV-ALK5 or LV-con-ALK5 injection at 24 h and 14 d after I/R (n = 6 biological replicates). *P < 0.05, compared to the LV-con-RNAi or LV-con-ALK5 group at the same time point (Student’s t test)
Fig 5: LK5 promotes BDA-positive fibers crossing the midline in the corpus callosum and red nucleus; ALK5 mediates dendritic plasticity after I/R.A a Representative images of BDA-positive fibers crossing the midline of the corpus callosum in each group (n = 5 biological replicates). b Representative images of BDA-positive corticorubral fibers crossing the midline to the right red nucleus in each group (n = 5 biological replicates). Arrows show the positive fibers. (scale bar = 100 µm). c Immunohistochemical analysis for BDA-positive fibers crossing the midline of the corpus callosum. d Immunohistochemical analysis for BDA-positive corticorubral fibers crossing the midline. *P < 0.05, compared to the LV-con-RNAi group; ?P < 0.05, compared to the LV-con-ALK5 group; #P < 0.05, compared to the sham group (ANOVA). e Representative images of Golgi–Cox pyramidal neurons in each group 24 h and 14 d after I/R (n = 5 biological replicates). (scale bar = 100 µm). f Comparison of the number of intersection points in Sholl analysis for Golgi–Cox pyramidal neurons. *P < 0.05, compared to the LV-con-RNAi group at the same time point; ?P < 0.05, compared to the LV-con-ALK5 group at the same time point; #P < 0.05, compared to the sham group at the same time point (ANOVA)
Supplier Page from MilliporeSigma for Anti-TGF β Receptor I antibody produced in rabbit