Phospho-Smad2/3 (Thr8) Antibody - #AF3367

Figure 4 FOXC2 driving EMT changes in PMCs may be achieved through the TGF-β1-Smad2/3 signaling feedback loop. A,B) Western blot analyses of FOXC2, Smad2/3, and p-Smad2/3 protein levels among different groups. n = 3 per group. Compared with the vector group, **p < 0.01, *p < 0.05. Compared with the vector group in the presence of TGF-β1, #p < 0.05. C,D) Western blot analyses of Smad2/3 and p-Smad2/3 protein levels among different groups. n = 3 per group. Compared with the vehicle group, **p < 0.01. Compared with the FOXC2-KD group, ##p < 0.01. E) Immunofluorescence staining images of nuclear accumulation of TGF-β1-induced Smad2/3 (red) in FOXC2-KD PMCs. Scale bar = 10 µm. F,G) Western blot analyses of Smad2/3 protein abundance in the cytoplasm and nucleus among different groups. n = 3 per group. Compared with the vehicle group, **p < 0.01. Compared with the TGF-β1, ##p < 0.01. H) The cell viability of PMCs treated with different concentrations of DGM (5, 10, 20, 40, 80, and 160 µM) for 24 h by CCK8 assay. n = 12 per group. Compared with the vehicle group, **p < 0.01. I) The cell viability of PMCs treated with TGF-β1 and different concentrations of DGM (10, 20, and 40 µM) for 24 h by CCK8 assay. n = 10 per group. Compared with the vehicle group, **p < 0.01. Compared with the TGF-β1 group, ##p < 0.01. J) Western blot analyses of the protein abundance of FOXC2, α-SMA, E-cadherin, Smad2/3, and p-Smad2/3 in PMCs treated with TGF-β1 and different concentrations of DGM (10, 20, and 40 µM) for 24 h. K–M) qRT-PCR analysis of mRNA expressions of FOXC2, α-SMA, and E-cadherin in PMCs treated with TGF-β1 and different concentrations of DGM (10, 20, and 40 µM) for 24 h. n = 6 per group. Compared with the vehicle group, **p < 0.01. Compared with the TGF-β1 group, ##p < 0.01, #p < 0.05.

Figure 6. SCs promote the migration of fibroblasts and keratinocytes. (A) In vitro wound-healing assays of 3T3 cells cultured in mediums from 3T3, RSC96 or S16 cells. (B) Migration rates were evaluated according to the closure area at 18 h after lesion induction. (C) In vitro wound-healing assays of HaCaT cells cultured in mediums from HaCaT, RSC96 or S16 cells. (D) Quantification of wound closure at 18 h after scratching. (E) In vitro wound-healing assays of 3T3 cells cultured in medium from RSC96 or S16 cells after control or Tgfb3 RNAi (siCtrl or siTgfb3) treatment. (F) Quantification of wound closure of 3T3 cells under different conditions. (G) In vitro wound-healing assays of HaCaT cells cultured under the indicated conditions. (H) Quantification of wound closure in HaCaT cells under the indicated conditions. (I) Immunoblot analysis of the expression of aSMA, phosphorylated Smad2/3 (p-Smad2/3) and Smad2/3 in 3T3 or HaCaT cells after incubation with mediums from RSC96 or S16 cells for 24 h. The images presented are representative of three independent experiments. Scale bars: a, c, e, g, 200 mm. Unpaired t test. The data are presented as the mean ± SEM, *P

Fig. 8 The potential role of the TGFβ1/SMAD pathway in the knockout of eIF3a to relieve EndMT. A, B In vitro experiment, western blot and the statistical results of TGFβ1 and p-SMAD2/3 proteins. C–E in vivo experiment, western blot and the statistical results of TGFβ1 and p-SMAD2/3 proteins. F–L in vivo experiment, western blot and the statistical results of TGFβ1 and p-SMAD2/3 proteins. Data are represented as the mean ± SD, n = 6–10

Fig. 8. Neferine inhibited the TGF-β1/Smad2/3 signaling pathway in abdominal aortic tissues SHRs. IHC analysis was performed to determine the protein expression of TGF-β1 (A), Smad2/3 and (B) p-Smad2/3 (C). All micrographs were taken at 400 × magnification. Scale bar = 50 µm. Representative images are shown on the left panel and statistical graph on the right panel. Data were denoted as means ± standard deviations; # p 

Figure 3 BMP8B triggers SMAD2/3 signaling to suppress adipogenesis. (A,B) Analysis using immunoblotting and quantification was conducted to assess the protein levels of p-SMAD1/5/8, p-SMAD2/3, p-ERK1/2, p-p38 MAPK, and p-JNK in LV-Bmp8b. (C) A model of BMPs-associated signal transduction. (D) Quantification was performed to determine the luciferase reporter activity driven by BRE, which pCMV-Bmp8b cotransfected with pCMV-Alk2, pCMV-Alk3, pCMV-Bmpr2, pCMV-Acrv2a, respectively. (E) Quantification was performed to determine the luciferase reporter activity driven by CAGA, which pCMV-Bmp8b cotransfected with pCMV-Alk2, pCMV-Alk4, pCMV-Alk5, pCMV-Alk7, pCMV-Tgfβr2, pCMV-Acrv2a, and pCMV-Acrv2b, respectively. (F,G) In the presence of DMH1 or TP0427736 HCL, the cells were induced to differentiate into adipocytes. On Day 8, Oil Red O staining was performed (F). Quantification of lipid content after adipogenic differentiation (G). Scale bar = 20 µm. The symbols in the charts represent three biological replicates. The data were presented as mean ± SD and analyzed using one-way ANOVA (ns not significant, ** p < 0.01, *** p < 0.001).

Fig. 5 Bmp8a activates Smad2/3 signaling to inhibit adipocyte differentiation in 3T3-L1 cells. a–d Representative western blot analysis and quantification of changes in p-Smad1/5/8, p-Smad2/3, p-ERK1/2, p-p38 MAPK, and p-JNK expression in LV-bmp8a cells (a, b) or LV-Bmp8a cells (c, d). Protein expression levels were quantified using ImageJ software and normalized to the amount of total protein (n = 3). e, f Representative Oil Red O staining photographs of LV-bmp8a and LV-Bmp8a 3T3-L1 cells were induced to adipogenic in the presence of DMH1 or TP0427736 HCL, dimethylsulfoxide (DMSO) as a vehicle and subjected to OD492 quantifications (n = 3). Scale bar = 20 µm. g Schematic diagram of BMP8 mediated signal transduction. BMP8 can activate Smad1/5/8 signal transduction through the receptor complex formed by type I receptor ALK2, ALK3, or ALK6 and type II receptor ACVR2A or BMPR2. Meanwhile, BMP8 can also activate Smad2/3 signal transduction through the receptor complex formed by type I receptors ALK4 or ALK5 and type II receptors ACVR2A, ACVR2B, or TGFBR2. h Non-expression of mouse Alk6 gene in 3T3-L1 cells (n = 3). i The qPCR quantification of the type I receptor (Alk2, Alk3, Alk4, Alk5, Alk7) and type II receptor (Acvr2a, Acvr2b, Bmpr2, Tgrβr2) transcripts expressed in 3T3-L1 cells (n = 3). j, k Quantification of the activity of BRE-driven luciferase reporters with pCMV-bmp8a (j) or pCMV-Bmp8a (k) cotransfected with pCMV-Alk2, pCMV-Alk3, pCMV-Bmpr2, pCMV-Acrv2a, respectively (n = 3). Renilla luciferase was used as the internal control. l, m Quantification of the activity of CAGA-driven luciferase reporters with pCMV-bmp8a (l) or pCMV-Bmp8a (m) cotransfected with pCMV-Alk2, pCMV-Alk3, pCMV-Bmpr2, and pCMV-Acrv2a, respectively (n = 3). Renilla luciferase was used as the internal control. Data were representative of at least three independent experiments. Data were analyzed by One-way ANOVA and presented as mean ± SD