Supplementary Figure 5: The effect of ABL1 variants (isoform 1a) on phosphorylation. | Nature Genetics

Supplementary Figure 5: The effect of ABL1 variants (isoform 1a) on phosphorylation.

From: Germline mutations in ABL1 cause an autosomal dominant syndrome characterized by congenital heart defects and skeletal malformations

Supplementary Figure 5

(a) Overall phosphotyrosine levels and phosphorylation of specific ABL1 substrates were analyzed by transiently expressing the wild-type and mutant constructs in HEK 293T cells and immunoblotting. Cells expressing both variants showed increased overall phosphotyrosine levels and phosphorylation of STAT5 when compared with wild-type cells. Increased levels of phosphorylated ABL1 were observed for Ala337Thr but not Tyr226Cys owing to the substitution of the Tyr226 residue, which is recognized by the anti-phospho-ABL1 antibody. No significant difference in the phosphorylation levels of CrKL, SMAD2, and SMAD3 was observed between cells expressing the mutants and wild-type protein. Antibodies used in detection included anti-phosphotyrosine (p-Tyr; 4G10) antibody for the overall phosphotyrosine level and anti-phospho-ABL1 (p-ABL1), anti-phospho-STAT5 (p-STAT5), anti-phospho-CrkL (p-CrkL), and anti-phospho-SMAD2 and SMAD3 (p-Smad2/3) antibodies to determine the phosphorylation levels of specific ABL1 substrates in whole-cell lysates. The level of GAPDH is used as an internal loading control. Experiments for each construct were performed in triplicate. (b) Quantification of the immunoblot results. Data are normalized to GAPDH protein levels, with the wild-type protein set at 1.0. **P ≤ 0.01, ****P ≤ 0.0001; n.s., P > 0.05.

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