Figure 7.
Figure 7. HSP70 and p53 expression level analysis. (A) Study of HSP70, p53, phosphorylated (Ser15) p53 protein expression level compared with actin in normal erythroid cells from CD34+ primary cells at day 7 of the erythroid culture. Cells have been treated with nutlin 3A, a p53 activator, which increased p53 stabilization, for 1 hour (1H) to 4 hours (4H) compared with nutlin 3B, the inactive enantiomer. In addition, these erythroid cells have been treated with (right western blot) or not (left western blot) MG132 proteasome inhibitor at 10 µM for 1 hour. Different concentrations of nutlin 3A and 3B (5 to 10 µM) for 1 hour have been also tested with (right western blot) or not (left western blot) MG132 proteasome inhibitor at 10 µM for 1 hour (western blots below). (B) Immunoprecipitation of HSP70 revealed by an antibody against p53 and HSP70 as a control in depleted RPL11 (shRPL11) UT7-EPO dependent erythroid cells, compared with the control (shSCR) after or not a MG132 treatment. IgG SCR lane: IgG control antibody in shSCR infected cells; HSP70 SCR: immunoprecipitation with a specific antibody for HSP70 in shSCR infected cells; IgG shRPL11: IgG control antibody in RPL11 depleted cells; HSP70 shRPL11: immunoprecipitation with a specific antibody for HSP70 in RPL11 depleted cells; IgG shRPL11+MG132: IgG control antibody on shRPL11 depleted erythroid cells after treatment with MG132; HSP70 shRPL11+MG132: immunoprecipitation with a specific antibody for HSP70 in RPL11 depleted cells, treated with MG132. The expression level analysis of HSP70 and p53 has been performed in total cell lysate (INPUT lanes) in order to validate that the p53 and HSP70 were correctly expressed in the samples before immunoprecipitation. (C) Study of p53, phosphorylated (Ser15) p53 protein expression compare with actin in depleted RPS19, RPL5, or RPL11 erythroid cells from CD34+ primary cells at day 7 of erythroid culture after a rescue or not with the lentivirus containing the WT HSP70 cDNA. Quantification of P-p53 compared with β-actin (bottom panel) has been performed on Image J, Java Image processing program (NIH Image).

HSP70 and p53 expression level analysis. (A) Study of HSP70, p53, phosphorylated (Ser15) p53 protein expression level compared with actin in normal erythroid cells from CD34+ primary cells at day 7 of the erythroid culture. Cells have been treated with nutlin 3A, a p53 activator, which increased p53 stabilization, for 1 hour (1H) to 4 hours (4H) compared with nutlin 3B, the inactive enantiomer. In addition, these erythroid cells have been treated with (right western blot) or not (left western blot) MG132 proteasome inhibitor at 10 µM for 1 hour. Different concentrations of nutlin 3A and 3B (5 to 10 µM) for 1 hour have been also tested with (right western blot) or not (left western blot) MG132 proteasome inhibitor at 10 µM for 1 hour (western blots below). (B) Immunoprecipitation of HSP70 revealed by an antibody against p53 and HSP70 as a control in depleted RPL11 (shRPL11) UT7-EPO dependent erythroid cells, compared with the control (shSCR) after or not a MG132 treatment. IgG SCR lane: IgG control antibody in shSCR infected cells; HSP70 SCR: immunoprecipitation with a specific antibody for HSP70 in shSCR infected cells; IgG shRPL11: IgG control antibody in RPL11 depleted cells; HSP70 shRPL11: immunoprecipitation with a specific antibody for HSP70 in RPL11 depleted cells; IgG shRPL11+MG132: IgG control antibody on shRPL11 depleted erythroid cells after treatment with MG132; HSP70 shRPL11+MG132: immunoprecipitation with a specific antibody for HSP70 in RPL11 depleted cells, treated with MG132. The expression level analysis of HSP70 and p53 has been performed in total cell lysate (INPUT lanes) in order to validate that the p53 and HSP70 were correctly expressed in the samples before immunoprecipitation. (C) Study of p53, phosphorylated (Ser15) p53 protein expression compare with actin in depleted RPS19, RPL5, or RPL11 erythroid cells from CD34+ primary cells at day 7 of erythroid culture after a rescue or not with the lentivirus containing the WT HSP70 cDNA. Quantification of P-p53 compared with β-actin (bottom panel) has been performed on Image J, Java Image processing program (NIH Image).

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