Journal: Scientific reports

Article Title: Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-β3 signaling in tenogenic differentiation.

doi: 10.1038/s41598-024-60717-z

Figure Lengend Snippet: Figure 4. Gene expression and Western blot of Smad2/3. Cells cultured as monolayer or on collagen matrix were incubated with 10 ng/ml TGF-β3 and/or 10 μM Y-27632. (A) Box plots represent the relative gene expression of Smad2/3. Asterisks mark differences between the stimulation groups with p < 0.05. Letters mark differences with p < 0.05 between collagen matrix and monolayer within same stimulation groups or in comparison to monolayer control (n = 4). (B) Representative western blot result of carboxy- terminal phosphorylation at Ser465/467 after 24 h stimulation. (C) Quantitative analysis of carboxy-terminal phosphorylation at Ser465/467 at 60 min, 12 h (n = 3) and 24 h (n = 5). Box plots represent the ratio of P-Smad to Smad2/3 and dots the values of the different donors.

Article Snippet: After blocking with 5% milk or BSA, depending on the respective antibody, in a Tris–buffered saline 0.5% Tween (TBST) solution overnight at 4 °C, membranes were stained with antibodies against total-smad 2/3 (1:1000, D7G7, #8685, Cell Signaling Technology), p-smad2 S245/250/255 (1:1000, #3104, Cell Signaling Technology), p-smad S465/467 (1:1000, E8F3R, #18338, Cell Signaling Technology) or p-smad3 S204 (1:500, A24906, Bioworld Technology) for 1 h. Then membranes were washed with TBST three times, incubated with an anti-rabbit HRP-conjugated secondary antibody (1:200, #7074, Cell Signaling Technology) for 1 h and washed again four times.

Techniques: Gene Expression, Western Blot, Cell Culture, Incubation, Comparison, Control, Phospho-proteomics

Journal: Scientific reports

Article Title: Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-β3 signaling in tenogenic differentiation.

doi: 10.1038/s41598-024-60717-z

Figure Lengend Snippet: Figure 5. Western blot results of Smad2/3 linker phosphorylation. Cells cultured as monolayer or on collagen matrix were incubated with 10 ng/ml TGF-β3 and/or 10 μM Y-27632. Representative Western blot result of linker phosphorylation at Ser245/250/255 of Smad2 after 24 h (A) and 30 min (B) stimulation and at Ser204 of Smad3 after 30 min stimulation (C). Quantitative analysis of linker phosphorylation at Ser245/250/255 (B: n = 5; D: n = 3) and Ser204 (C, n = 4). Box plots represent the ratio of P-Smad to Smad2/3 and dots the values of the different donors.

Article Snippet: After blocking with 5% milk or BSA, depending on the respective antibody, in a Tris–buffered saline 0.5% Tween (TBST) solution overnight at 4 °C, membranes were stained with antibodies against total-smad 2/3 (1:1000, D7G7, #8685, Cell Signaling Technology), p-smad2 S245/250/255 (1:1000, #3104, Cell Signaling Technology), p-smad S465/467 (1:1000, E8F3R, #18338, Cell Signaling Technology) or p-smad3 S204 (1:500, A24906, Bioworld Technology) for 1 h. Then membranes were washed with TBST three times, incubated with an anti-rabbit HRP-conjugated secondary antibody (1:200, #7074, Cell Signaling Technology) for 1 h and washed again four times.

Techniques: Western Blot, Phospho-proteomics, Cell Culture, Incubation

Journal: Heliyon

Article Title: Mangiferin inhibits chronic stress-induced tumor growth in colorectal liver metastases via WAVE2 signaling pathway

doi: 10.1016/j.heliyon.2023.e13753

Figure Lengend Snippet: MGF inhibits activation of HSCs via the WAVE2 signaling pathway. (A) Transduced HSCs treated with TGF-1 (2.5 ng/mL) and IF for SMA show that MGF consistently suppresses TGF-1-induced myofibroblast differentiation. (B) Following 24 h of serum starvation and treatment with TGF-1, control and MGF-treated HSCs are shown. Western blot analysis for detection of HSC activation markers, α-SMA and p -SMAD2, and activator β2-AR and TGF-β1. Data are presented as the mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The stock solution was used to prepare different concentrations in the test media. including β2-AR polyclonal antibody (Cat. #8513, CST), WAVE2 polyclonal antibody (Cat. # YT4898, ImmunoWay), VEGF polyclonal antibody (Cat. # sc-7269, Santa Cruz Biotechnology), α-smooth muscle actin (α-SMA) (Abcam, Cat. # ab5694), a polyclonal antibody against p -SMAD2 (Cat. #3104, CST) at a 1:1000 and GAPDH mouse monoclonal antibody (Cat. # ab8245, Abcam) at 1:5000 dilution.

Techniques: Activation Assay, Control, Western Blot