Computational protocol: Smooth muscle cell-specific Tgfbr1 deficiency promotes aortic aneurysm formation by stimulating multiple signaling events

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Protocol publication

[…] ATA segments were homogenized in 50 mM Tris-HCl buffer supplemented with phenylmethanesulfonyl fluoride (0.1 μM), leupeptin (10.0 nM), and a phosphatase inhibitor cocktail (Invitrogen). The concentration of the protein solution was determined with the BCA protein assay kit (Thermo Scientific, 23225). Total protein (5 μg) was separated with SDS-PAGE gels, and membranes were blotted with primary antibodies purchased from R&D (Tgfbr2, MAB532), Santa Cruz (Tgfbr1, SC398), and Cell Signaling (SMAD2, 5339; SMAD3, 9523; pSMAD3, 9520; ERK1/2, 4696, and others as detailed above). Immunoblots were developed with Lumigen substrate (#TMA-6) and imaged with x-ray films. The intensity of the band of interest was quantified with ImageJ software, and data were normalized to β-actin or total protein of the assayed molecules. [...] SigmaPlot version 13.0 was used for statistical analyses. Student t-tests, Fisher’s exact tests, Chi-square tests, one-way ANOVA, two-way repeated measurement ANOVA, and Kaplan Meier survival analyses were performed as appropriate, with P < 0.05 considered to be statistically significant. Datasets were evaluated using normality and equivalence variance testing. For those failing this evaluation, logarithmic and exponential transformations were employed to meet these requirements. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Aneurysm, Deficiency Diseases