Figure 4

The transcription factor MITF is expressed in progenitor cells, leucocytes and primary moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or IL-10 (10 ng/mL). For the analysis of CD34⁺ progenitor and blood cells, cell-type specific total RNA was used. qRT-PCR analysis: (A, B) relative level of MITF and (C, D) GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (E) MITF protein level and phosphorylation status was analyzed by western blotting in two different donors (lanes 1–4 and lanes 5–7, respectively). Phosphorylated MITF was detected by mobility shift (slower migrating band at 70 kDa). Western blotting revealed an additional, slower migrating band that supposedly represents the phosphorylated protein of higher molecular weight of approximately 70 kDa. “+ phosphatase”: cell lysates were incubated with phosphatase. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.