Ab18528

Proteins were run in 10% SDS-Poly acrylamide gel and transferred to nitrocellulose membrane using a Trans-Blot Turbo System-Bio-Rad (only when LC3 II protein was analyzed, a 12% SDS-Poly acrylamide gel was used). To evaluate samples from lysosomal isolation and cell lysates, 10 μg to total protein was loaded on the gel. The nitrocellulose membrane was incubated with a blocking solution (TBS 0.1% Tween-20, 5% BSA) for 1 h. First antibodies were diluted in a TBS 0.1% Tween-20, 2% BSA solution and incubated overnight at 4°C. After three washes, the membrane was incubated with secondary antibodies in a TBS 0.1% Tween-20 solution for 1–2 h at room temperature. After three washes, the membrane was developed in an Odyssey-FC system (Licor). Antibodies and their working solutions were the follow: anti-TDP-43 (Proteintech #10782-2-AP, 1:2,000), anti-Lamp2A (Abcam #ab18528, 1:2,000) and anti-GAPDH (Santa Cruz #sc-365062, 1:5,000), anti-Hsc70 (Thermo Scientific #13D3, 1:5,000), anti-Flag (Sigma-Aldrich M2, 1:1,000), anti-LC3 A/B (Cell Signaling #4108, 1:1,000), p62 (Abnova #H00008878-M01, 1:5,000), BAG3 (Novus Biological # NBP2-27398ss, 1:2,000).

Immunohistochemistry was performed with LAMP-2A (1:100, Abcam, ab18528), N-CoR (1:100, Santa Cruz, sc-1609) for cells and N-CoR (1:100, Novus, CO, US, NBP1-28,863) for tissues. After being equilibrated at − 20 °C for 15 min, brain tumor tissues were sectioned at a range of 5–7 μm and fixed with 4 °C pre-cold acetone. Cultured cells growing on glass slides were fixed with 4% of formaldehyde. The tissue or cell sections were incubated with 3% of H₂O₂ for 10 min, followed by 10% of BSA as blocking solution for 1 h at room temperature. Then the sections were incubated with the primary antibodies overnight at 4 °C and hybridized with horseradish peroxidase labeled secondary antibody (Beyotime, China) for 1 h at room temperature. The diaminobenzidine kit (DAB kit; Long Island Biotec, Shanghai, China) was applied for visualization and hematoxylin (BASO, China) for counterstain.