Figure 1: Conditional expression of myc-tagged hFUS from the MAPT locus reveals increased stability of ALS mutant protein.

(a) Organization of the endogenous (τ) and targeted (τ^OFF) MAPT locus on mouse Chromosome 11. An hFUS expression cassette (light blue) was introduced into exon 2 of MAPT, replacing the endogenous start codon. Excision of the LOX-STOP-LOX transcriptional stop sequence by Cre-mediated recombination allows expression of the myc-tagged hFUS^WT, hFUS^R521C and hFUS^P525L cDNAs from the τ^ON allele of the MAPT locus. (b) Southern blot analysis of targeted ES cell clones following BamHI digestion and hybridization with a 5′ external probe (green, a). Wild-type ES cells (left, +/+) display a 9.0-kb band. Targeted ES cells (right, +/τ^OFF) yielded a 3.3-kb band corresponding to the τ^OFF allele in addition to the 9.0-kb band corresponding to the untargeted allele at the MAPT locus. (c) RT–qPCR analysis of hFUS transcripts in total brain and total spinal cord (p90, N=4). Data are represented as mean and s.e.m. (d) Western blot analysis of total spinal cord, brain and sciatic nerve extracts. 7x myc-tagged hFUS migrated at a higher molecular weight (90 kDa) relative to endogenous mouse FUS (73 kDa). Immunoblotting with anti-FUS antibody did not detect myc-hFUS. All mutants analysed were heterozygous for the τ^ONhFUS alleles (with the exception of Supplementary Fig. 4A–C) and tau expression was maintained in these animals. (e) Immunoprecipitation of P90 mouse spinal cord extracts with anti-myc or anti-FUS antibodies. myc-hFUS bands were detected with an anti-FUS antibody following immunoprecipitation with an anti-myc antibody. (f) Western blot analysis of cyclohexamide (CHX)-treated embryonic stem cell-derived motor neurons (ESMNs). At 16 h post CHX treatment, myc-hFUS was no longer detected with anti-myc antibody in cells derived from τ^ONhFUS^WT animals but persisted in τ^ONhFUS^R521C and τ^ONhFUS^P525L samples. Levels of endogenous mouse FUS detected with anti-FUS antibody were similarly decreased in all samples. CyclinB1 was used as an internal positive control for CHX treatment efficacy. (g) Quantification of myc-hFUS levels, normalized to myc-hFUS levels at 0 h, in CHX-treated ESMNs. Data are represented as mean and s.e.m. of N=3 differentiations.