Figure 6.
Figure 6. SENP1 desumoylase activity plays a critical role in GATA1 desumoylation and activation. (A) A representative blot showing that non-CMS cells show significantly higher levels of sumoylated GATA1. The sumoylated form of GATA1 was determined by IP with anti-SUMO followed by Western blotting with anti-GATA1. The ratios of SUMO-GATA1/GATA1 were quantified. IB, immunoblot. (B) Non-CMS cells show a significantly high SUMO-GATA1/GATA-1 ratio as compared with CMS. P < 0.05; Student’s t test. The results are the summary for n = 4 for both CMS and non-CMS groups. Each bar represents the mean, and error bars represent SE. For each sample, the levels were measured in triplicates. Each experiment was repeated three times. (C) Fused SUMO-GATA1 cannot restore the remarkable CMS phenotype in a background of GATA1 KO. Non-CMS had a similar result. Each bar represents a mean of three replicates, and the experiment was repeated at least twice. (D) Western blot analysis confirming significantly higher levels of SUMO-fused Gata1 in the overexpressed (OE) line. Lane 1 represents CMS cells lines overexpressing fused SUMO-GATA1 in the GATA1 KO background. Please note the significantly higher levels of SUMO-GATA1 in the GATA1 KO background. Lane 2 shows the relative levels of SUMO-GATA1 and GATA1 in non-CMS cells. Lane 3 shows the relative levels of SUMO-GATA1 and GATA1 in CMS cells. (E) GATAK137R (GATA1KR) mutant causes the polycythemic phenotype in both CMS and non-CMS subjects under hypoxia. Each bar represents a mean of three replicates, and the experiment has been repeated at least twice. (F) Western blot analysis showing overexpression of GATA1K137R in the non-CMS cells. Lane 1 represents non-CMS cell lines overexpressing GATA1K137R in the GATA1KO background. Lane 2 shows the relative levels GATA1 in uninfected CMS cells. Lane 3 shows the relative levels of GATA1 in uninfected non-CMS cells. (C–F) Each bar represents the mean of three replicates, and the experiment was repeated at least twice. Error bars represent SEM. **, P < 0.01.

SENP1 desumoylase activity plays a critical role in GATA1 desumoylation and activation. (A) A representative blot showing that non-CMS cells show significantly higher levels of sumoylated GATA1. The sumoylated form of GATA1 was determined by IP with anti-SUMO followed by Western blotting with anti-GATA1. The ratios of SUMO-GATA1/GATA1 were quantified. IB, immunoblot. (B) Non-CMS cells show a significantly high SUMO-GATA1/GATA-1 ratio as compared with CMS. P < 0.05; Student’s t test. The results are the summary for n = 4 for both CMS and non-CMS groups. Each bar represents the mean, and error bars represent SE. For each sample, the levels were measured in triplicates. Each experiment was repeated three times. (C) Fused SUMO-GATA1 cannot restore the remarkable CMS phenotype in a background of GATA1 KO. Non-CMS had a similar result. Each bar represents a mean of three replicates, and the experiment was repeated at least twice. (D) Western blot analysis confirming significantly higher levels of SUMO-fused Gata1 in the overexpressed (OE) line. Lane 1 represents CMS cells lines overexpressing fused SUMO-GATA1 in the GATA1 KO background. Please note the significantly higher levels of SUMO-GATA1 in the GATA1 KO background. Lane 2 shows the relative levels of SUMO-GATA1 and GATA1 in non-CMS cells. Lane 3 shows the relative levels of SUMO-GATA1 and GATA1 in CMS cells. (E) GATAK137R (GATA1KR) mutant causes the polycythemic phenotype in both CMS and non-CMS subjects under hypoxia. Each bar represents a mean of three replicates, and the experiment has been repeated at least twice. (F) Western blot analysis showing overexpression of GATA1K137R in the non-CMS cells. Lane 1 represents non-CMS cell lines overexpressing GATA1K137R in the GATA1KO background. Lane 2 shows the relative levels GATA1 in uninfected CMS cells. Lane 3 shows the relative levels of GATA1 in uninfected non-CMS cells. (C–F) Each bar represents the mean of three replicates, and the experiment was repeated at least twice. Error bars represent SEM. **, P < 0.01.

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