Supplementary Figure 6: The effect of the ABL1 Tyr245Phe variant (isoform 1b) on phosphorylation. | Nature Genetics

Supplementary Figure 6: The effect of the ABL1 Tyr245Phe variant (isoform 1b) on phosphorylation.

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

Supplementary Figure 6

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 the Tyr245Phe variant showed decreased overall phosphotyrosine levels when compared with cells expressing the wild-type protein. Phosphorylated ABL1 was not observed for Tyr245Cys and Tyr245Phe owing to the substitution of the Tyr245 residue, which is recognized by the anti-phospho-ABL1 antibody. 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), and anti-phospho-CrkL (p-CrkL) 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. Longer exposure of the blot is also included to show the overall phosphotyrosine patterns more clearly.

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