Ab181838

Protein was extracted in modified RIPA buffer (50mM Tris-HCl, 1% Nonidet P-40 substitute, 150mM NaCl, 1mM EDTA, and 1% glycerol supplemented with 20μg/mL each aprotinin, pepstatin, leupeptin, and PMSF) through glass-bead disruption, separated by PAGE, and blotted onto a PVDF membrane. Immunodetection was performed using anti-Procaspase 3 1:1000 (ab32499, Abcam), for TDP43-RFP, anti-RFP 1:2000 (M155-3; MBL Medical Biological Laboratories Co.), and anti–β-tubulin 1:25 (E7, Developmental Hybridoma Ban, Iowa State University) primary antibodies. Secondary antibodies were as follows, goat anti-Rb-HRP 1:10,000 (170-6515, BioRad) to detect Pro-caspase 3, and goat anti-Ms-HRP 1:10,000 (170-6516, Bio-Rad) to detect TDP43-RFP or tubulin were applied.
For PHB2 co-immunoprecipitation (co-IP), proteins were extracted in IP buffer (20mM HEPES pH7.4, 2mM MgCl2, 300mM NaCl, 0.1% Tween-20 supplemented with 20μg/mL each aprotinin, pepstatin, leupeptin, and PMSF) through glass-bead disruption. Co-IP was accomplished using 500μg of total protein extract (volumes normalized) with 5μL anti-TDP43 (ab190963, Abcam) and 50μL anti-Rb Dynabeads (11203D, Invitrogen). Following separation by PAGE, and blotting onto a PVDF membrane, immunodetection of PHB2 was performed using anti-REA 1:1000 (ab181838, Abcam) primary antibody and secondary antibody goat anti-Rb-HRP 1:10,000 (170-6515, Bio-Rad).

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.