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. 2006 Jul 6;25(14):3264–3274. doi: 10.1038/sj.emboj.7601228

Figure 7.

Figure 7

Regulation of GATA-1 ubiquitination by SCF and erythropoietin. (A) GATA-1 ubiquitination is increased in response to signalling by SCF and erythropoietin. BM-SCF or BM-Epo cells were starved of cytokine for 6 h. SCF or erythropoietin was then re-administered and GATA-1 immunoprecipitated at the times shown. GATA-1 ubiquitination was analysed with an anti-ubiquitin antibody and GATA-1 levels with an anti-GATA-1 antibody (M-20; middle panel). Sixty minutes after induction, GATA-1 levels increased, most likely owing to new GATA-1 synthesis. Phosphorylated GATA-1 (P-GATA-1) is indicated. LT indicates cells grown continuously (long term) in SCF. β-Tubulin levels verify that equivalent cell numbers were used (lower panel). (B) Phosphorylation is required for SCF-induced GATA-1 turnover. BM-SCF cells that express FLAG-tagged GATA-1 mutated in individual phosphorylation sites (S26A, S49A and S72A) were induced with SCF. Immunoblotting with anti-FLAG antibody showed that SCF did not cause a phosphorylation shift or loss of GATA-1. FLAG-tagged wild-type GATA-1 is lost like the endogenous protein (compare lower panel and panel A middle panel). (C) Inhibition of phosphorylation prevents GATA-1 ubiquitination. BM-SCF cells were starved of growth factor for 6 h. SCF was then re-administered either in the absence (SCF) or presence (SCF+U0126) of the MEK inhibitor U0126. Samples were taken at the times indicated, immunoprecipitated with anti-GATA-1 antibody and the level of ubiquitination determined via Western blotting using an anti-ubiquitin antibody. The middle panel shows GATA-1 levels following re-probing of the blot with a second anti-GATA-1 antibody (M-20). Phosphorylated and non-phosphorylated GATA-1 are indicated by asterisks. (D) Acetylation is required for GATA-1 turnover. Upper panel: GATA-1 acetylation mutant is not degraded in response to SCF signalling; endogenous GATA-1 in the same cell is lost (second panel from top). Fourth panel: Acetylated GATA-1 (Ac-GATA-1) is degraded faster than bulk GATA-1 (lower panel, where the same blot was re-probed) upon mitogen signalling.