Figure 1. Aggregation state of NM-HA in mouse neuroblastoma cell populations.
(A) Immunofluorescence staining of mouse N2a neuroblastoma cells stably expressing soluble yeast Sup35 prion domain NM tagged with the HA antibody epitope (NM-HAsol) and N2a subclones 1c, 2e, and 3b persistently producing NM-HA aggregates (NM-HAagg). NM was detected using mAb anti-HA (green), and nuclei were stained with Hoechst (blue). Scale bar: 5 μm. Note that individual cell clones differ in their respective NM-HA expression levels (Krammer et al, 2009). Cell clones had been isolated from a N2a NM-HA bulk population exposed to recombinant NM amyloid fibrils. (B) The presence of SDS-resistant NM-HA in N2a subclones was determined by a filter trap assay. NM was detected using mAb anti-HA. (C) SDD–AGE analysis of lysates from N2a cells expressing NM-HAsol and NM-HAagg. NM-HA was detected using mAb anti-HA. (D) Schematic illustration of NM-HA. A region highly abundant in Q/N at the amino-terminus is shown in blue (residues 1–38). Note that also other regions in N are enriched in Q/N. The octapepetide repeat region is depicted in green, and the carboxyterminal region of N is marked in black. Chymotrypsin preferentially cleaves at the carboxyl side of amide bonds of tyrosine, tryptophan, and phenylalanine (symbolized by arrow heads). Note that the M domain lacks these residues. Three tyrosine residues are present in the HA epitope. Putative fragments that correspond to the length of peptides identified by Western blot are indicated. (E, F) NM-HA aggregates derived from different N2a cell clones exhibit comparable chymotrypsin patterns. Lysates of N2a NM-HAsol and N2a NM-HAagg clones 1c, 2e, and 3b were subjected to increasing amounts of chymotrypsin or left untreated. Proteins were analyzed by SDS–PAGE and Western blot. NM was detected by 4A5 anti-M domain antibody raised against an epitope spanning amino acid residues 229–247 (Krammer et al, 2008b) (E) or mAb anti-HA (F) (both antibody-binding sites indicated in (D)).