rabbit anti total smad  (Novus Biologicals)

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

Journal: International Journal of Molecular Sciences

Article Title: Lithium Treatment Improves Cardiac Dysfunction in Rats Deprived of Rapid Eye Movement Sleep

doi: 10.3390/ijms231911226

Figure Lengend Snippet: Transforming growth factor beta (TGF-β) signaling pathway in REM sleep deprivation with and without lithium treatment. The REM sleep-deprived hearts ( n = 5) had greater expression levels of TGF-β, phosphorylated Smad 2/3, and α-smooth muscle actin (α-SMA) compared with the control ( n = 5) and lithium-treated REM sleep-deprived ( n = 5) hearts. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Blots were probed with the following antibodies against regulator proteins involved in cardiac fibrogenesis: TGF-β (1:2000, polyclonal, #3711; Cell Signaling Technology, Beverly, MA, USA), TGF-β RI (1:1000, polyclonal, #sc-398; Santa Cruz Biotechnology, Santa Cruz, CA, USA), total Smad 2/3 (1:2000, monoclonal, #8685; Cell Signaling Technology), phosphorylated Smad 2/3 (1:2000, monoclonal, #8828; Cell Signaling Technology), α-SMA (1:5000, monoclonal, #ab7817; Abcam, Cambridge, UK), AGTR1 (1:2000, polyclonal, #AAR-011; Alomone Labs, Jerusalem, Israel), total NF-κB p65 (1:2000, monoclonal, #8242; Cell Signaling Technology), phosphorylated NF-κB p65 (1:2000, monoclonal, #3033; Cell Signaling Technology), Orai1 (1:2000, polyclonal, #4281; ProSci Incorporated, Poway, CA, USA), STIM1 (1:2000, monoclonal, #610954; BD Transduction Laboratories, San Diego, CA, USA), TRPC 1 channel (1:2000, polyclonal, #ACC-010; Alomone Labs), TRPC3 (1:5000, polyclonal, #ACC-016; Alomone Labs), and TRPC6 (1:2000, polyclonal, #ACC-017; Alomone Labs).

Techniques: Expressing