Phospho-Smad3 (Ser423+Ser425) Antibody - #AF8315

Fig. 6. TGFBR1 overexpression partly abolishes Rg3's inhibition on CFs growth, collagen synthesis, together with Smads activation. (A) CFs were infected with recombinant adenovirus for 48 h, and later stimulated by TGF-β1 (10 ng/ml) and Rg3 (20 μM), and Edu assay was performed to detect CFs proliferation (magnification, 200 × ). Red and blue fluorescence indicate proliferating cells as well as nuclei, separately. (B) Edu-positive cell proportion. (C) Expression of proliferation and collagen-related proteins in CFs following Ad-TGFBR1 or control adenovirus transfection. (D) Protein expression of TGFBR1 signaling in CFs after transfection. Relative PCNA (E), CDK6 (F), Cyclin D1 (G), collagen I (H), collagen III (I), p-TGFBR1 (J), p-Smad2 (K), and p-Smad3 (L) expression. Data are represented by mean ± SD for at least 3 groups. ∗p < 0.05, ∗∗p < 0.01. n.s, not significant.

Figure 3. Regorafenib down-regulates TGF-β1/Smad and TGF-β1/non-Smad signals in pulmonary fibroblasts. (A) CAGAmouse embryonic fibroblast (NIH-3T3) cells were exposed to TGF-β1 (5 ng/mL) or a series concentration (0–32 µM) in serum-free medium for 18 h; (B) Mlg cells were treated with TGF-β1 (5 ng/mL) and/or RG (2 µM, 4 µM) for 30 min, and Western blot was used to detect Smad3, Smad2, and their phosphorylation expression levels. Densitometric analysis are shown beside; (C) Mlg cells were incubated with RG (2 µM, 4 µM) and/or TGF-β1 (5 ng/mL) for 12 h to analyze the Erk1/2 and Akt and their phosphorylation levels by Western blotting. Densitometric analysis are shown beside; (D) BLM-PPF cells were incubated with RG (2 µM, 4 µM) for 12 h to analyze Erk1/2 and Akt and their phosphorylation levels by Western blotting. Densitometric analysis are shown beside. β-tubulin or GAPDH were used as a loading control. Data in (A–D) are means ± standard error of mean (SEM); ### p < 0.001, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA), NS: not significant.

FIGURE 5 Liver fibrosis‐related protein expressions under Robo2 knockdown in LX‐2 cells (A, B) Western blot analysis showed the protein expressions of Robo2, α‐SMA, collagen I, p‐Smad2, p‐Smad3, Samd2/3, p‐PI3K, PI3K, p‐Akt and Akt in sTREM‐1+Lv‐shNC and sTREM‐1+Lv‐shRobo2 groups of LX‐2 cells. * p < 0.05

Figure 9 TFPI2 promotes TGF-β/Smad signaling activation. Human renal glomerular endothelial cells (hRGECs) were infected with adenovirus encoding shRNA targeting TFPI2 (TFPI2 shRNA) or overexpressing TFPI2 (TFPI2 OE), followed by stimulation of 5 ng/ml TGF-β2 for 48 h. A, the expression of SMAD7, TGFBR1, TGFBR2, SMAD2/3, and phospho-SMAD2/3 (p-SMAD2/3) was determined by Western blot. Semiquantitative analysis of (B) SMAD7, (C) TGFBR1, and (D) TGFBR2, as well as (E and F) the ratio of p-SMAD2/3 to SMAD2/3. G and H, immunofluorescent staining of SMAD2/3 in hRGECs. Yellow arrows indicated nuclear translocation of SMAD2/3. Data are shown as the mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. TGF-β, transforming growth factor beta; TFP12, tissue factor pathway inhibitor 2.