Quantification of 11kDa and NS1 expression during B19V infection of CD36⁺ EPCs. (A) Quantification of B19V 11kDa- and NS1-encoding mRNAs. CD36⁺ EPCs were infected with B19V. At 24, 48, and 72 hours after infection, total RNA was isolated, treated with DNase, reverse-transcribed, and quantified for absolute copies of mRNA by multiplex real-time PCR for NS1-mRNA/β-actin-mRNA and 11kDa-mRNA/β-actin-mRNA as described in “RT real-time PCR.” The copy numbers of the 11kDa- and NS1-encoding mRNAs were normalized by copy numbers of β-actin mRNA in the same reaction and presented as numbers per copy of β-actin mRNA. (B) Purity of purified fusion proteins GST-NS1(aa1-181) and GST-11kDa. Purified GST-NS1(aa1-181) and GST-11kDa proteins were resolved in SDS-PAGE 10% gel and stained with Coomassie blue as shown. (C-D) Quantification of the steady-state protein level of 11kDa versus NS1 during B19V infection. GST-NS1(aa1-181) (100 ng) and GST-11kDa (100 ng) as seen in panel B and a serial dilution of them as shown were loaded in SDS-PAGE 8% and SDS-PAGE 15% for Western blot (C lanes 1-7 and D, respectively). At 48 hours after infection, 1.5 × 10⁵ of CD36⁺ EPCs with or without (mock) B19V infection were harvested, directly dissolved in SDS lysis buffer, and loaded in lanes 6-7 (SDS-PAGE 8%), lanes 8-9 (SDS-PAGE 6%) (C), and lanes 3-4 (SDS-PAGE 15%) (D). Results from lanes as indicated were also quantified with Quantity One software (GE Healthcare) and plotted to the right in panels C-D. Arrow and arrowhead in panel C show NS1-specific band and GST-NS1(aa1-181), respectively; and arrow and arrowhead in panel D show 11kDa-specific band and GST-11kDa, respectively. CD36⁺ indicates CD36⁺ EPCs.