Figure A1: Increased ubiquitin levels in TDP-43M337V mice but hTDP-43 itself is not ubiquitinated. Human TDP-43 was immunoprecipitated from brain homogenates derived from nontransgenic and homozygous TDP-43m337v mice. Briefly, brain homogenates containing 500 μg protein were incubated with 1.5 μg mouse monoclonal TDP-43 antibody overnight at 4°C with gentle shaking. Protein G agarose was added for 4 h at 4°C then pelleted by centrifugation. Protein thus captured was eluted using sample loading buffer and resolved by SDS/PAGE for Western blot analysis. Shown are immunoblots of the inputs and the immunoprecipitated proteins, probed using an antibody to ubiquitin or to total TDP-43. Note that a marked increase in ubiquitin levels is observed in the homogenates derived from homozygous mice prior to immunoprecipitation. Nonetheless, the immunoprecipitated human TDP-43 is not immunopositive for ubiquitin. Arrow = IgG Heavy Chain.