Fig. 4

hnRNPF and hnRNPH1/2 localize with SMN and PLS3 in fibroblasts and motor neuron processes. Murine embryonic fibroblasts’ (MEFs) cell cultures were derived from Smn ^+/−; PLS3V5 ^tg/tg embryos and stained with V5 for PLS3 transgene; hnRNP-H1/2, hnRNP-F, and F-actin with phalloidin. a PLS3V5 localized to the same region as hnRNP F. b hnRNP F similarly localized with SMN. c hnRNP H1/2 localized to the same region with tagged PLS3. d hnRNP H1/2 localized to the same region with SMN. e SMN localized with PLS3 also in mouse spinal motor neurons processes in primary culture. Motor neurons were derived from GFP-Hb9-expressing embryos, which labels motor neurons. Endogenous protein was detected using immunohistochemistry. Quantitative analysis of cellular localization using confocal images of processes reports the percentage of localization for SMN fluorescence within processes. Protein fluorescence criterion was manually set using controls within the COLOMBUS software (Perkin Elmer), see the “Methods” section for details. f Localization of SMN with hnRNP F was seen using the same methods as described in e. g Localization of SMN with hnRNP H1/2 was seen using the same methods as described in e. Protein localization assays were done as three independent trials where each sample in each trial was a merge of images from multiple wells on a 96-well plate. Images were taken using an automated program, and then, slices were chosen incorporating planes with the nuclei