primary antibodies against phopho-smad2/3 (psmad2/3, 1:1000)  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: MODULATION OF COLLAGEN-BINDING INTEGRINS AFFECTS FIBROBLAST ACTIVATION AND INHIBITS FIBROSIS

doi: 10.1101/2025.05.14.653428

Figure Lengend Snippet: (A) TGF-β mRNA and active TGF-β, but not total TGF-β (which includes the matrix-bound TGF-β) were increased in livers from Alb-cKO animals. N=22/25 for mRNA, 20/19 for TGF-β protein. *p<0.05. (B-C) TGF-β signaling is enhanced in Alb-cKO livers as evidenced by western blot analysis showing increased pSMAD2, 3, 4 (in C) and decrease in the inhibitor SMAD7 (in C). N=8/9 for pSMAD2, 10/12 for pSMAD3, 4/4 for pSMAD4, and 15/20 for SMAD7. *p<0.05. (D) Evaluation of Alb-cKO hepatocytes confirms the increase in TGF-β mRNA and protein. N=6/6 for mRNA, 45/35 for active TGF-β and 16/15 for total TGF-β. *p<0.05, **p<0.01. (E) Caveolin-1 is diminished both in Alb-cKO livers and hepatocytes. N=11/14 for the livers and 7/8 for the hepatocytes. *p<0.05. (F) Isolated hepatocytes maintained for 45 min without serum show a decrease in baseline pFAK expression compared to control (CT) hepatocytes. N=16/15. **p<0.005. (G) Inhibition of FAK in cultured wildtype hepatocytes leads to a rise in TGF-β mRNA expression. Hepatocytes were cultured on vitronectin and treated for 24 hours with 5μM of the inhibitor PF573228 (Tocris). N=9/4. *p<0.05. (H) Hepatic stellate cells from Alb-cKO animals show no decrease in β1 integrin, and no change in TGF-β mRNA expression. N=7/7 for β1 integrin, N=9/16 for TGF-β. *p<0.05. (I-J) In Mx-cKO animals, β1 integrin is deleted in hepatocytes (I) and in hepatic stellate cells (J), TGF-β mRNA expression is increased in both cell types. Western for β1 integrin: N=3/3 for hepatocytes and 4/4 for hepatic stellate cells, qPCR: N=22/28 for hepatocytes and 12/11 for stellate cells. **p<0.005, ****p<0.0001. (K) mRNA and protein expression of α-SMA were increased in livers from Alb-cKO animals compared to littermate controls. More αSMA-stained cells were found in Alb-cKO liver sections. Bars represent 100μm. N=20/19 for mRNA, 14/14 for protein and 19/20 for histology sections. Livers were obtained from 16-week-old animals, hepatocytes were isolated from 8-10-week-old animals and hepatic stellate cells from 20-24-week-old mice. Data were analyzed using unpaired t-tests.

Article Snippet: Western blotting for collagen-type-I (Santa cruz, 28654), α-SMA (Sigma-Aldrich A2547), integrin α10 (Thermofisher, PA5-67829), integrin α11 (Thermo scientific PA5-23897, Bio-techne AF6498, R&D MAB4235), integrin β1 (Millipore, #MAB1997), caveolin-1 (BD 610406), pFAK/FAK (Cell signaling 3284/Millipore 06-543), pAKT/AKT (Cell signaling #9271/9272), pERK/ERK (Cell signaling 4376/9102), pSMAD2/SMAD2 (Cell signaling 3108/5339), pSMAD3/SMAD3 (Cell signaling 9520/9513), pSMAD4/SMAD4 (Abgent 3251a, Epitomics 1676-1), and pSMAD7/SMAD7 (Thermo scientific 42-0400) were performed and adjusted to GAPDH (Sigma-Aldrich) or HPRT (Invitrogen, #PA5-106984).

Techniques: Western Blot, Isolation, Expressing, Control, Inhibition, Cell Culture, Staining

Journal: Stem Cell Research & Therapy

Article Title: Subchondral injection of human umbilical cord mesenchymal stem cells ameliorates knee osteoarthritis by inhibiting osteoblast apoptosis and TGF-beta activity

doi: 10.1186/s13287-025-04366-7

Figure Lengend Snippet: UCMSCs inhibited both the osteoblast apoptosis and the Smad2-dependent TGF-β signaling pathway in OA. ( A ) Representative images of TUNEL staining. Scale bar, 100 μm. ( B ) Quantitative analysis of the TUNEL positive cells. ( C - E ) IHC staining and quantitative analysis of TGFβ1 and pSmad2. Scale bar, 100 μm. Data presented as means ± SEM by one-way ANOVA with Turkey’s post hoc test. ** P < 0.01 and *** P < 0.001

Article Snippet: After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated with the primary antibodies against beta actin (β-actin; Proteintech, 20536-1-AP; 1:10000), alkaline phosphatase (Alp; Abcam, Arigo, ARG57422 ; 1:5000), runt-related transcription factor-2 (Runx2; Huabio, ET1612-47; 1:5000), Phospho-SMAD2 (pSmad2; Thermofisher, 44-244G; 1:1000) and Smad2 (CST, 5339 S; 1:1000) overnight at 4 °C.

Techniques: TUNEL Assay, Staining, Immunohistochemistry

Journal: Stem Cell Research & Therapy

Article Title: Subchondral injection of human umbilical cord mesenchymal stem cells ameliorates knee osteoarthritis by inhibiting osteoblast apoptosis and TGF-beta activity

doi: 10.1186/s13287-025-04366-7

Figure Lengend Snippet: UCMSC-CM inhibited osteoblast apoptosis and TGF-β pathway of MC3T3-E1 cells. (A-B) TUNEL staining and quantitative analysis of MC3T3-E1 cells treated with TNFα and UCMSC-CM. Scale bar, 100 μm. (C-D) Flow cytometry analysis of MC3T3-E1cells after treatment with TNFα and UCMSC-CM. UL: necrosis cells, UR: late apoptotic cells, LL: viable cells and LR: early apoptotic cells. (E-F) The protein expression of p-Smad2 in MC3T3-E1 cells treated with TNFα and UCMSC-CM. Data presented as means ± SEM by one-way ANOVA with Turkey’s post hoc test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated with the primary antibodies against beta actin (β-actin; Proteintech, 20536-1-AP; 1:10000), alkaline phosphatase (Alp; Abcam, Arigo, ARG57422 ; 1:5000), runt-related transcription factor-2 (Runx2; Huabio, ET1612-47; 1:5000), Phospho-SMAD2 (pSmad2; Thermofisher, 44-244G; 1:1000) and Smad2 (CST, 5339 S; 1:1000) overnight at 4 °C.

Techniques: TUNEL Assay, Staining, Flow Cytometry, Expressing