Skip to main content
. 2020 Oct 22;48(20):11785–11798. doi: 10.1093/nar/gkaa908

Figure 2.

Figure 2.

NANPs show enrichment in membrane bound compartments and cytosolic compartments. Microglia were transfected with 5 nM Cy3 labeled NANPs and at 2- and 4-h post transfection, whole cell lysates (WCL) were fractioned into cytosolic (Cytosolic Isolation Buffer, CIB), membrane/organelle (Membrane/organelle isolation buffer, MIB) and nuclear/cytoskeleton (Nuclear/cytoskeleton isolation buffer, NIB) fractions. (A) Fractions were evaluated for protein expression of GAPDH, COX IV, histone 3 (H3), Rab7, and Actin using immunoblot analysis. (B) Schematic of cell fractionation protocol. Fluorescence (Ex540/Em580) of cell fractions was evaluated using a SpectraMax iD5 plate reader from molecular devices at (C) 2 h and (E) 4 h. The DNA-Cy3 strand used to compose all DcD, DcR and Dc2′F is 1.74 ± 0.005 times less fluorescent than the RNA-Cy3 strand used to compose RcR, RcD, Rc2′F; therefore, the fluorescent signal of DcD, DcR, and Dc2′F in each fraction was multiplied by 1.74 to compensate for the lower fluorescent signal observed with the DNA-Cy3 strand. Data are represented as the mean ± (SEM) for a minimum of three independent experimental replicates. Asterisks indicate statistical significance compared to cells transfected with DcD NANPs and pound symbols indicate statistical significance compared to cells transfected with RcD NANPs (one-way ANOVA, P-value < 0.05). The ratio of the CIB fraction to the MIB fraction is displayed at (D) 2 h and (F) 4 h.