NF-L sequence in 3′UTR of transgene affects the disposition of GFP-tagged NF-L protein in transfected Neuro2a cells. A, Schematic diagram of transgenes with (pGFP/NF+3′UTR) or without (pGFP/NF–3′UTR) NF-L 3′UTR sequence (thick red line) in the 3′UTR of pEGFP/C2 expression vector. Transgenes contain identical GFP/NF-L coding sequence (open green–red lines), stop codon (angled arrows), and flanking 5′UTR and 3′UTR sequences (thin lines). B, C, Confocal microscopy of GFP fluorescence (B) and anti-NF-L reactivity (C) showing diffuse distribution of GFP-tagged NF-L protein expressed from pGFP/NF–3′UTR transgene. D, E, GFP-tagged NF-L protein in short, curvilinear filamentous arrays from low-level expression of pGFP/NF+3′UTR. F, G, GFP-tagged NF-L protein in multiple punctate aggregates from higher-level expression of pGFP/NF+3′UTR. H, I, GFP-tagged NF-L protein in large juxtanuclear aggregates from high level of pGFP/NF+3′UTR expression. J, Anti-GFP immunoblot of GFP-tagged NF-L protein (97 kDa) from cells transfected with pGFP/NF–3′UTR (–) or pGFP/NF+3′UTR (+) in total lysates (total lysate) or lysates separated by centrifugation into supernatant and pelleted fractions showing proteins in pelleted fractions solubilized in buffers of increasing solvent strength, including 1% Triton X-100 (triton-soluble), 2 m urea (2M urea-soluble), 4 m urea (4M urea-soluble), and 2% SDS (SDS-soluble). Immunoblots were conducted on 1:30 aliquots of Triton X-100-soluble fractions, 1:5 aliquots of 2 and 4 m urea-soluble fractions, and 1:2 aliquots of SDS-soluble fractions. K, Quantitation of anti-GFP immunoblot of from cells transfected with pGFP/NF–3′UTR (open bars) or pGFP/NF+3′UTR (filled bars) in total lysates (total lysate) or in fractions solubilized in 1% Triton X-100 (triton-soluble), 2 m urea (2M urea-soluble), 4 m urea (4M urea-soluble), and 2% SDS (SDS-soluble). t test; ***,^###p < 0.001. Scale bars, 10 μm.