Rabbit anti lrrc47

HEK293T cells were transfected with GFP and AP(50)-GFP lentiviral constructs in 10 cm dishes according to our transfection protocol described above. Cells were lysed in 500 μl of ice-cold Pierce IP lysis buffer (ThermoFisher, cat. no. 87787) containing 1x protease inhibitor cocktail (Sigma, cat. no. 4693132001) 5 days post-transfection. Lysates were incubated on ice for 1 h with periodic mixing by inversion and subsequently centrifuged at 14,000 g for 15 min at 4°C. The resulting supernatants were transferred to new Eppendorf tubes and the co-IP was performed following the manufacturers protocol for the SDS elution using the GFP-trap magnetic particles M-270 kit (Chromotek, cat. no. gtdk-20). Briefly, 200 μL of cell lysate was diluted with 300 μL of the provided dilution buffer and 25 μL of equilibrated GFP-trap magnetic beads was added. The solution was incubated overnight at 4°C with end-over-end rotation. Next, the beads were washed three times with the provided wash buffer, resuspended in 80 μL of 2x SDS-sample buffer, boiled for 5 min at 95°C, and analyzed via Western Blot using anti-GFP (Aves, cat. no. GFP-1010, 1:1000) and rabbit anti-LRRC47 (Proteintech, cat. no. 23217–1-AP, 1:500). LRRC47 protein detected was endogenous LRRC47. Images were acquired using an LI-COR Odyssey CLx.

MACs-sorted PSA-NCAM+ neural stem and progenitor cells from healthy controls and C9-FTD/ALS patients were collected in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Roche). Protein quantity was measured by the BCA assay (Pierce) and samples were run on a 10% SDS gel at room temperature in with compatible loading buffers for the LI-COR Odyssey CLx. The gel was transferred onto a nitrocellulose membrane (VWR) via a semi-dry transfer. The membrane was blocked with intercept blocking buffer (Licor, cat. no. 927–60010) for 1 h at room temperature, incubated with primary antibodies overnight at 4°C, washed three times with 0.1% PBS-T, then incubated with the appropriate IRDye (Licor, 1:5000) for 1 h at room temperature. After five washes with PBS, blots were visualized using an LI-COR Odyssey CLx. Sample signals were normalized based on the revert 700 total protein stain (Licor). The following primary antibodies were used: rabbit anti-LRRC47 (Proteintech, cat. no. 23217–1-AP, 1:500), chicken anti-GFP (Aves, cat. no. GFP-1010, 1:1000), rabbit anti-C9ORF72 (Proteintech, cat. no. 22637–1-AP, 1:500), and ribosomal protein S6 (Thermofisher, cat. no. MA5–15123, 1:500).