Cac tip ptd m01

Mouse brain tissue was lysed in RIPA lysis buffer (Cell signaling), supplemented with protease inhibitors cocktail and phosphor inhibitor (Thermo Fisher Scientific), and kept on ice for 30 mins. The brain homogenate was then ultracentrifuged at 50,000g for 15 mins at 4°C. The supernatant was transferred to a new tube as RIPA-soluble protein fraction. The pellet was homogenized in 10% sucrose buffer before being ultracentrifuged at 50,000g for 15 mins at 4°C. After ultracentrifuge, the pellet was discarded and the supernatant was transferred to a new tube and incubated with sarkosyl (Sodium lauroyl sarcosinate) at a final concentration of 1% for 1 hour at 37°C. Following incubation, the samples were ultracentrifuged at 50,000g for 30 mins at 4°C. The supernatant was discarded and the pellet was re-suspended to obtain the insoluble protein fraction. Soluble and insoluble fractions were then boiled in SDS-PAGE loading buffer for immunoblotting. Blots were probed with antibodies for mouse anti-hTDP-43 (1:1000, Proteintech, 60019–2-Ig), rabbit anti-total TDP-43 (1:1000, Proteintech, 10782–2-AP), mouse anti-human TDP-43, phospho Ser409/410 (1:2000 , COSMOBIO, CAC-TIP-PTD-M01), rabbit anti-GFP (1:1000, Cell signaling, 2956s) and mouse anti-GAPDH (1:5000, Santa Cruz, sc-32233).