Fig 2: Smcr8 CRISPR F0 mice developed signs of autoimmunity. (A) Anti-dsDNA (dsDNA) autoantibody concentration in the plasma of wild-type control or Smcr8 CRISPR F0 mice at 150, 200, and 250 d old. (B) Heat map of plasma IgM activity against 124 different autoantigens in age-matched C9orf72+/+ (n = 3), C9orf72+/− (n = 4), C9orf72−/− (n = 2), and Smcr8 CRISPR F0 (n = 15) mice. (C) T-cell clonality in the spleens of Smcr8 CRISPR F0 mice and C9orf72+/+, C9orf72+/−, and C9orf72−/− mice by PCR amplification of the somatic T-cell receptor β (TCRβ) locus. LN2 and LN3 represent ES cell lines derived from lymph node T cells harboring monoclonal TCRβ. (D) Flow cytometry analysis of total cell number of each of the indicated cell populations in the spleens of age-matched wild-type control, C9orf72−/−, and Smcr8 CRISPR F0 mice. n = 4. (n.s.) Not significant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001 by two-way ANOVA with Dunnett's multiple comparisons.

Fig 3: Evidence that SMCR8 protein is poly-ubiquitinated. a The FL-SMCR8 construct was transfected in 293T cells and immunoprecipitated with α-FLAG antibody-bound agarose. A Western blot of whole cell lysates probed with α-FLAG antibody shows expression of full-length FL-SMCR8 protein plus HMW products consistent with PTMs (left). Probing with α-UBB antibody marks HMW products in immunoprecipitates consistent with either poly-ubiquitinated FL-SMCR8 protein or the presence of other HMW ubiquitinated proteins that co-IP with the SMCR8 complex (right). IP reactions were in the presence or absence of 50 μg/ml RNases. b C-terminal V5-tagged SMCR8 and empty vector or HA-tagged ubiquitin were coexpressed in 293T cells and treated or not treated with the proteasome inhibitor MG132. Expression of SMCR8-V5 protein and empty vector, in the presence but not absence of MG132, produces HMW bands on Western blots that are consistent with post-translational modification of SMCR8 at multiple sites. SMCR8-V5 protein coexpressed with HA-UBB and without MG132 shows the same HMW bands, which increase in signal intensity upon incubation with MG132. c V5- or HA-epitope-tagged SMCR8 was coexpressed with empty vector or FLAG-tagged UBB in 293T cells and incubated overnight in the presence or absence of MG132. Cell lysates were subjected to immunoprecipitation with α-FLAG agarose, followed by Western blotting and probing with α-HA (top left panel), α-V5 (top right) or α-FLAG (bottom left) antibodies. A HMW smear seen in immunoprecipitates is consistent with poly-ubiquitination of tagged SMCR8 proteins. In general, overexpression of ubiquitin does not lead to a significant decrease in full-length SMCR8 protein levels

Fig 4: C9ORF72–SMCR8–WDR41 form a protein complex. (A) Illustration of the gene-editing strategy for making C9ORF72-HA stable expression hES cell lines. Zinc finger nuclease was used to introduce a double-strand break in the AAVS1 locus. The puromycin resistance selection gene and CAGGS-driven C9ORF72-HA expression cassette are stably incorporated into the AAVS1 locus through homologous recombination. (B) Identification of protein coimmunoprecipitated with the C9-long protein isoform by quantitative mass spectrometry. The red dots represent proteins significantly enriched in samples with C9-long-HA expression after anti-HA immunoprecipitation with an adjusted P-value < 0.05 by moderated t-test. (C) Differentially expressed proteins in bone marrow-derived macrophages (BMDMs) from C9orf72+/+ and C9orf7−/− mice by quantitative mass spectrometry. n = 2 for each genotype. The red dots represent differentially expressed proteins with an adjusted P-value of <0.05 by moderated t-test. (D–F) HEK293 cells were left untreated or were treated with control siRED or siRNA against C9ORF72 as indicated. (D) qPCR validation of C9ORF72 siRNA knockdown efficiency and SMCR8 expression level with the indicated treatment. (E) Quantification of SMCR8 protein expression normalized to GAPDH by Western blot. For both D and E, n = 3. (*) P < 0.05; (**) P < 0.01 by two-tailed Student's t-test. Error bar represents standard deviation. (F) Representative Western blot image for endogenous SMCR8 and GAPDH with the indicated siRNA treatment.

Fig 5: Analysis of 16 C9ORF72 antibodies by immunoprecipitation.(A) HEK-293 cell lysates were prepared and immunoprecipitation was performed using the indicated C9ORF72 antibodies pre-coupled to protein G-Sepharose (prot. G). Controls included the Protein G-Sepharose alone incubated with cell lysate or protein G-Sepharose pre-coupled with the antibodies but incubated with lysis buffer. Samples were washed and processed for immunoblot with C9ORF72 antibody PT22637. (B) HEK-293 cell lysates were prepared and immunoprecipitation was performed using the indicated C9ORF72 antibodies pre-coupled to protein A-Sepharose (prot. (A). Controls included the Protein S-Sepharose alone incubated with cell lysate or protein A-Sepharose pre-coupled with the antibodies but incubated with lysis buffer. Samples were washed and processed for immunoblot with C9ORF72 antibody GTX634482. (C) Lysates were prepared from HEK-293 cells, parental (+/+) and KO (-/-) and immunoprecipitation was performed using C9ORF72 antibody GTX632041 pre-coupled to protein G-Sepharose. Samples were washed and processed for immunoblot with C9ORF72 antibody PT22637. Blots were also performed for SMCR8 and WDR41. The Ponceau stained transfer of the blot is shown as a protein loading control. Aliquots (10%) of the cell lysates before (starting material, SM) and after incubation with the antibody-coupled beads (unbound, UB) were processed in parallel. (D) Table indicating the total spectrum count and protein identification probabilities for the indicated gene products proteins obtained by mass spectrometry analysis of the immunoprecipitated samples from three independent experiments (D1–D2–D3).