Anti tdp 43 polyclonal

Yeast were grown and induced in galactose containing medium for 5h from overnight cultures supplemented with raffinose. For plasmids under the control of the HSE promoter, cells were grown as for the thermotolerance assay and harvested after the incubation at 37ºC. Cultures were normalized to A_600nm = 0.6, 3ml cells were harvested, treated in 0.1M NaOH for 5 min at room temperature, and cell pellets were then resuspended into 1x SDS sample buffer and boiled for 4min. Lysates were cleared by centrifugation at 14,000 rpm for 2min and then separated by SDS-PAGE (4–20% gradient, BioRad), and transferred to a PVDF membrane. Membranes were blocked in Odyssey Blocking Buffer (LI-COR). Primary antibody incubations were performed at 4ºC overnight. Antibodies used: anti-GFP monoclonal (Roche Applied Science), anti-TDP-43 polyclonal (Proteintech), anti-FUS polyclonal (Bethyl Laboratories), anti-Hsp104 polyclonal (Enzo Life Sciences), and anti-PGK monoclonal (Invitrogen). Membranes were imaged using a LI-COR Odyssey FC Imaging system.

TDP-43, p65 and tau were quantified using Western immunoblot. Proteins (15 μg/sample) were heated at 95°C for 5 minutes in SDS sample buffer. For immunoprecipitation study, anti-TDP-43 polyclonal (ProteinTech, Chicago) or anti-p65 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz) was bound to protein G-coated magnetic beads (Dyanl, Invitrogen, Camarillo) and was incubated with 50 μg of lysate overnight at 4°C. After washing, immunoprecipitates were eluted with SDS sample buffer. Samples were resolved by 10% SDS-PAGE and transferred to a PVDF membrane (Polyscreen, PerkinElmer, Boston, MA). The membrane was incubated with anti-p65 (Invitrogen, Camarillo) or anti-TDP-43 2E2-D3 antibody (Abnova, Walnut), and western blot image was obtained using a chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL). Each protein was estimated by standardization with actin.