Fig. 1.

p38 MAPK signaling regulates γ-globin transcription. (A) p38 MAPK mRNA levels were quantified by RT-qPCR analysis using gene specific primers (Table 1); non-targeting scrambled (Scr) siRNA was used as a control. Data were calculated as the mean ± standard error of the mean (SEM), p<0.05 was considered significant. WB analysis was performed using 150μg of total protein and an anti-total p38 (t-p38) antibody, actin was used as a loading control. (B) The effect of p38 MAPK silencing on Gγ-globin and Aγ-globin transcription were quantified using gene specific primers for RT-qPCR analysis. (C) Three stable lines including PC, kMKK3, and kMKK6 were established in K562 cells using the pcDNA, pcDNA3-MKK3 and pcDNA3-MKK6 expression vectors respectively and then clones were isolated (see “Experimental procedures”). WB was performed with 150μg of protein using an anti-HA antibody to confirm gene expression. Levels of phosphophyrolated-p38 (p-p38) and t-p38 were determined by WB. (D) The effect of stable enforced activation of p38 MAPK on γ-globin transcription was determined by RT-qPCR analysis. (E) ELISA was performed to quantify the effect of activating p38 MAPK on HbF expression (see “Experimental procedures”). HbF levels were normalized by total hemoglobin (t-Hb) and total protein (t-Protein). (F) HbF positive cells were visualized by immunostaining with FITC conjugated anti-γ-globin antibody (HbF-FITC); DAPI staining was performed to visualize cell nuclei (magnification ×400). At least 500 DAPI positive cells were counted along with the number of FITC positive cells in the same field and then used to calculate the percentage of HbF positive cells.