Ab104223

Following low serum induction of differentiation (post 48 hours), C2C12 cells and human primary myoblasts were treated with either DMSO (2μL/ml) as a negative control, z-DEVD-FMK (20μM), or a 3-hour pulse of PAC1 (25μM). Treatments were refreshed at four days post low serum induction of differentiation. After six days of differentiation conditions, cells were fixed with 4% PFA (in PBS) for 10min at RT, then treated with 0.3% Triton X100 (in PBS) for 10 min at RT. TDP43 was detected using anti-TDP43 1:500 (ab104223, Abcam) and PHB2 using anti-REA 1:250 (ab181838, Abcam). Secondary antibodies used were goat anti-mouse Alexa 568 1:2000 (A11031, Invitrogen) to detect TDP43, and goat anti-rabbit Alexa 488 1:2000 (A11008, Invitrogen) to detect PHB2. Cells were also co-stained with DAPI (20ng/ml in PBS) for 10min at RT. Coverslips were prepped for microscopy using DAKO mounting medium. The resulting fluorescence was detected by microscopy using a Zeiss Observer Z1, with a Pan Apochromat objective 20X, or 63X under oil immersion with a 0.63X camera adapter. TDP43 inclusions were counted visually.

Polyclonal rabbit anti-Fxr1p (13194-1-AP, Proteintech; dilution: 1:200), polyclonal rabbit anti-KIF1C (ab125903, Abcam; dilution: 1:500), polyclonal rabbit anti-Mbnl1 (kindly gifted by Maury Swanson; dilution: 1:1000), monoclonal rabbit anti-Telethonin (ab133646, Abcam; dilution: 1:1000), monoclonal mouse anti-TDP-43 (ab104223, Abcam; dilution: 1:500), monoclonal mouse anti-G3BP (ab56574, Abcam; dilution: 1:1000), monoclonal mouse anti-Alpha Tubulin (T8203, Sigma; dilution: 1:1000), monoclonal mouse anti-Puromycin (EQ0001, Kerafast; dilution: 1:1000), monoclonal mouse anti-Nuclear Pore Complex Proteins (mAb 414, kindly gifted by Maury Swanson; dilution: 1:1000), and polyclonal chicken anti-Alpha Tubulin (ab89984, abcam; dilution: 1:1000) were used for protein localization via IF, either alone or in combination with FISH.

iMNs plated on coverslips were fixed using 4% paraformaldehyde (PFA) for 15 min and blocked for 1 h with 3% bovine serum albumin (BSA) and 0.1% TritonX-100 in phosphate buffered saline PBS. iMNs were incubated with the following primary antibody: mouse anti-GRP75 (Abcam, ab2799, 1:200), rabbit anti-GRP75 (1:200, Abcam, ab53098), rabbit anti-BiP (Abcam, ab21685, 1:500), goat anti-ChAT (Millipore, AB144P, 1:500), chicken anti-MAP2 (Sigma Aldrich, AB15452, 1:500), rat anti Isl1-2 (DSHB 39.4D5), mouse anti-HB9 (1:500, DSHB 81.5C10), mouse anti-TDP43 (ABCAM ab104223, 1:500), in blocking buffer overnight at 4 °C. After three washes with PBS, cells were incubated in blocking buffer with appropriate Alexa Fluor secondary antibodies and DAPI for 1 h at RT, followed by three PBS washes and coverslips were mounted on glass slides. Confocal images were acquired with FluoViewTM FV1000 (Olympus) fitted with a 20 × or 40 × air objective and 60 × immersion oil objective.