anti total smad 2 3  (Cell Signaling Technology Inc)

Journal: Biomaterials Research

Article Title: Myofibroblast-Targeting Extracellular Vesicles: A Promising Platform for Cardiac Fibrosis Drug Delivery

doi: 10.34133/bmr.0179

Figure Lengend Snippet: αFAP-EL@CLD loaded with GW788388 protects against cardiac fibrosis. (A) Schematic illustration of the procedure used to produce αFAP-EL@CLD loaded with GW788388 (αFAP-EL@CLD: GW788388). (B) TEM images showing the shape and size of Con-EL@CLD: GW788388 and αFAP-EL@CLD: GW788388. (C) NTA indicating the size and size distribution of Con-EL@CLD: GW788388 and αFAP-EL@CLD: GW788388. (D) The cumulative release curves of GW788388 in Con-EL@CLD and αFAP-EL@CLD in FBS ( n = 3). (E to G) NRCFs were treated with 10 ng/ml TGF-β1 for 48 h, followed by treatment with 50 μg/ml αFAP-EL@CLD: GW788388 at 24 h. (E) The activation level of NRCFs was evaluated by detecting the fluorescence intensity of α-SMA at 48 h. (F) Quantitative PCR analysis of the mRNA levels of Acta2 and Col1α1 in each treatment group. (G) The phosphorylation of SMAD family member 3 (Smad3) was detected by Western blotting. (H) Histological analysis of mouse hearts after 15 d of treatment, using H&E staining, Masson’s trichrome staining, and Sirius Red staining to evaluate the degree of cardiac fibrosis.

Article Snippet: Anti-glyceraldehyde-3-phosphate (anti-GAPDH) (Cat. No. 97166), anti-total SMAD family member 3 (Smad3) (Cat. No. 9523P), anti-phospho-Smad3 (anti-pSmad3) (Cat. No. 9520P), and anti-β-tubulin (Cat. No. 86298) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Fluorescence, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Staining

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: 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 Ser 465/467 after 24 h stimulation. ( C ) Quantitative analysis of carboxy-terminal phosphorylation at Ser 465/467 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: 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 Ser 245/250/255 of Smad2 after 24 h ( A ) and 30 min ( B ) stimulation and at Ser 204 of Smad3 after 30 min stimulation ( C ). Quantitative analysis of linker phosphorylation at Ser 245/250/255 ( B : n = 5; D : n = 3) and Ser 204 ( 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