Delivery of anti-synuclein antibody H3C into primary neurons.
A, fluorescence microscopy images of primary neurons treated with
nanoparticles loaded with a monoclonal antibody H3C against α-synuclein
or (B) with H3C and Polysorbate 80 alone and stained with a Alexa
Fluor 594-conjugated secondary and co-stained with DAPI. C, Western
blot analysis of whole-cell lysates (W) and protein-G pulldown assays
(G) from neurons treated with H3C-NPs (lanes 3 and
4), empty nanoparticles (lane 5 and 6), or H3C-NPs
after preincubation with anti-LDL receptor antibody (lanes 7 and
8). Lane 1 = H3C antibody (50 ng), lane 2 =
recombinant human α-synuclein (50 ng). Blot was probed with a goat
antibody (SC7012) against N-terminal α-synuclein. D,
immunocytochemistry of neurons treated with H3C-NPs then fixed and stained
with SC7012, a goat antibody against the N terminus of α-synuclein. The
primary antibodies were detected using Alexa Fluor 488-conjugated anti-mouse
and Cy3-conjugated anti-goat, respectively. NP-delivered H3C colocalizes with
SC7012. Scale bar = 50 μm.