Fig. 3.
Fig. 3. The expression of cellular PrPC is substantially up-regulated during differentiation of monocytes to myeloid DCs. / Flow cytometric analysis of PrPC expression during the maturation of DCs (A) or macrophages (B). Adherent peripheral blood mononuclear cells (PBMCs) were differentiated to DCs (A) or macrophages (B) in vitro. PrPC surface expression was monitored by staining PrPC (monoclonal antibody 6H3) and subsequent flow cytometry at days 2, 5, 7, and 10 of cell culture. PrPC expression is progressively up-regulated on successive days (d2-d10) for DCs (A), but there is no substantive change for macrophages (B). Isotype controls are indicated by i. (C) Western blots confirm the substantive up-regulation of total cellular PrPC during differentiation of adherent PBMCs to DCs (left lanes, days 2, 5, 7, 10), but not in macrophages (right lanes, days 2, 5, 7, 10). Note that the high-molecular-weight species visible at day 10 of DC maturation (left-hand lanes) represents a minor band only visible at very high levels of expression. (D) Molecular weight species of PrPC in lysates prepared from cultured human DCs compared with lysates of tissue from human brain and human spleen. The protein loading has been adjusted to allow comparison of tissues. DCs and spleen contain equal protein loading; brain loading is 10% of the total protein level of splenic tissue (determined by scanning densitometry of Coomassie-stained gels). Myeloid DCs generated in vitro and spleen express similar glycoform species. (E) Agarose gel electrophoresis of RT-PCR products of the PrP gene. A 528-bp fragment of the gene coding for PrP was amplified by RT-PCR from messenger RNA from myeloid DCs (lane 2), LPS-stimulated myeloid DCs (lane 3), and monocytes (lanes 4 and 5). No amplification of thePrP gene in the same samples by Taq polymerase (lanes 6-9) shows that no DNA was present in these samples. Amplification of the PrP gene from genomic DNA is shown for positive controls (lanes 10 and 11). One-Kb standard ladders (Gibco-BRL) are shown (lanes 1 and 12).

The expression of cellular PrPC is substantially up-regulated during differentiation of monocytes to myeloid DCs.

Flow cytometric analysis of PrPC expression during the maturation of DCs (A) or macrophages (B). Adherent peripheral blood mononuclear cells (PBMCs) were differentiated to DCs (A) or macrophages (B) in vitro. PrPC surface expression was monitored by staining PrPC (monoclonal antibody 6H3) and subsequent flow cytometry at days 2, 5, 7, and 10 of cell culture. PrPC expression is progressively up-regulated on successive days (d2-d10) for DCs (A), but there is no substantive change for macrophages (B). Isotype controls are indicated by i. (C) Western blots confirm the substantive up-regulation of total cellular PrPC during differentiation of adherent PBMCs to DCs (left lanes, days 2, 5, 7, 10), but not in macrophages (right lanes, days 2, 5, 7, 10). Note that the high-molecular-weight species visible at day 10 of DC maturation (left-hand lanes) represents a minor band only visible at very high levels of expression. (D) Molecular weight species of PrPC in lysates prepared from cultured human DCs compared with lysates of tissue from human brain and human spleen. The protein loading has been adjusted to allow comparison of tissues. DCs and spleen contain equal protein loading; brain loading is 10% of the total protein level of splenic tissue (determined by scanning densitometry of Coomassie-stained gels). Myeloid DCs generated in vitro and spleen express similar glycoform species. (E) Agarose gel electrophoresis of RT-PCR products of the PrP gene. A 528-bp fragment of the gene coding for PrP was amplified by RT-PCR from messenger RNA from myeloid DCs (lane 2), LPS-stimulated myeloid DCs (lane 3), and monocytes (lanes 4 and 5). No amplification of thePrP gene in the same samples by Taq polymerase (lanes 6-9) shows that no DNA was present in these samples. Amplification of the PrP gene from genomic DNA is shown for positive controls (lanes 10 and 11). One-Kb standard ladders (Gibco-BRL) are shown (lanes 1 and 12).

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