Quantifying Phosphorylated SMAD3 Signaling

After isolation, content determination and electrophoresis, proteins were electroblotted [31 (link)] and incubated with a monoclonal rabbit anti-phosphorylated SMAD3 (1:1000; S423 + S425, EP823Y; Abcam), anti-SMAD3 (1:1000; EP568Y; Abcam), polyclonal rabbit anti-TGFβ-RI (1:200; Santa Cruz Biotechnology), anti-TGFβ-RII (1:200; Santa Cruz Biotechnology), mouse anti-VDR (1:100, D-6; Santa Cruz Biotechnology), anti-α-smooth muscle actin (α-SMA, 1:000 Dako Cytomatic, Glostrup, Denmark), anti-total tubulin and anti-GAPDH antibodies (Sigma Aldrich) following horseradish peroxidase conjugate goat anti-rabbit or anti-mouse IgGs (Pierce, Rockford, IL, USA). Specific complexes were revealed and quantified and densitometric blot analysis performed in three independent experiments [32 (link)].

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Campione E., Di Prete M., Costanza G., Saggini A., Agostinelli S., Terrinoni A., Centofanti F., Rapanotti M.C., Bianchi L., Ferlosio A., Scioli M.G, & Orlandi A. (2023). Increased Occurrence of Cutaneous Leiomyomas and Dermatofibromas in Patients with Uterine Leiomyomas without Fumarate Hydratase Gene Mutations. Dermatopathology, 10(3), 231-243.

Publication 2023

EP568Y anti-TGFβ-RI anti-TGFβ-RII

Actin Anti antibodies Anti iggs Densitometric Electrophoresis Gapdh Goat Horseradish peroxidase Isolation Mouse Proteins Rabbit Smad3 Smooth muscle Tgfβ Tubulin

Corresponding Organization : Catholic University Our Lady of Good Counsel

Top 5 similar protocols

Variable analysis

independent variables

Electrophoresis
Electroblotting

dependent variables

Phosphorylated SMAD3 protein levels
SMAD3 protein levels
TGF-β-RI protein levels
TGF-β-RII protein levels
VDR protein levels
α-SMA protein levels
Total tubulin protein levels
GAPDH protein levels

control variables

Antibody dilutions used for immunoblotting

controls

Positive control: None specified
Negative control: None specified

Annotations

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