Subcellular distribution of Smn and hnRNP R in isolated embryonic motoneurons.

<p>(A) Motoneurons showed reduced Smn protein levels upon lentiviral knockdown of Smn. Uninfected or GFP-infected mouse embryonic motoneurons were used as controls. Levels of calnexin and hnRNP R were not affected. For this experiment a C-terminal antibody directed against hnRNP R was used as reported recently <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110846#pone.0110846-Glinka1" target="_blank">[29]</a>. This antibody recognizes distinct hnRNP R isoforms. (B) Representative images of motoneurons cultured for 7DIV and labeled against Smn (scale bar: 10 µm). GFP-transfected controls revealed immunoreactive signals for Smn in the cytosol, in neuronal processes and in Gem-like nuclear structures. Upon lentiviral Smn knockdown both cytosolic Smn immunoreactivity (Uninfected set as ‘1’, n = 4, N = 51; GFP 1.02±0.04, n = 4, N = 60; sh-Smn 0.34±0.02, n = 4, N = 74; P<0.001, t = 19.19) and number of Gems per nucleus (Uninfected 1.03±0.18, n = 4, N = 51; GFP 0.97±0.15, n = 4, N = 60; sh-Smn 0.08±0.02, n = 4, N = 74; P<0.01, t = 4.929) were significantly reduced in comparison to uninfected cells. (C) Subcellular distribution of hnRNP R in soma, axon and growth cone of primary motoneurons cultured for 5DIV and costained against synaptophysin (SynPhys) and neurofilament (NF-L) (scale bar: 10 µm (upper row), 5 µm). (D) Lentiviral knockdown of hnRNP R led to a dose-dependent reduction of hnRNP R levels. Calnexin and Smn protein were not altered significantly. (E) HnRNP R knockdown was also detected by immunofluorescence validating the used antiserum peptide ICN 1-18 (GFP 1.00±0.04, n = 8, N = 100; sh-hnRNP R 0.48±0.04, n = 6, N = 63; P<0.0001, t = 8.719, DF = 12) (scale bar: 10 µm).