Mouse anti lamp1

For immunofluorescence assay, cells were grown in coverslips and transfected with siRNA or indicated plasmids. After 36–48 h transfection, cells were washed with PBS and fixed with 4% formaldehyde. The embryos then blocked with blocking solution (3% BSA, 0.3% Triton X-100 in PBS) for 30 min, and incubated with primary antibodies, including rabbit anti-mTOR (Cell Signaling Technology, 2983, 1:300), mouse anti-DSTYK (Santa Cruz, sc-374487, 1:100), mouse anti-LAMP1 (Santa Cruz, sc-20011, 1:100), rabbit anti-V5 (Proteintech, 14440-1-AP, 1:200), rabbit anti-RAB7A (Proteintech, 55469-1-AP, 1:200), rabbit anti-DSTYK (Proteintech, 20102-1-AP, 1:200), rabbit anti-TFEB (Proteintech, 13372-1-AP, 1:200), mouse anti-V5 (Abcam, ab27671, 1:300), overnight at 4 °C. Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, A21206, 1:500), Alexa Fluor 568 donkey anti-rabbit IgG (Invitrogen, A10042, 1:500) and Alexa Fluor 594 donkey anti-mouse IgG (Invitrogen, A21203, 1:500) were used as a secondary antibody. Immunofluorescence staining was imaged with LSM880NLO confocal microscope with a ×63 oil objective (Carl Zeiss).

Protein samples were mixed with an equivalent volume of 2X Novex sample buffer (Life Technologies) supplemented to 5% β-mercaptoethanol. Proteins were denatured by incubating at room temperature for 30 min or by heating at 95 °C for 1–5 min before loading into SDS-polyacrylamide gels (Life Technologies). Proteins were transferred to Immobilon membranes (Millipore, Billerica, MA) and immunoblotted with the primary antibody at 4 °C overnight. The next day, blots were incubated with an HRP-conjugated secondary antibody (Promega, Madison, WI) and bands were detected by enhanced chemiluminescence using Western Lightning Plus-ECL reagents (Perkin Elmer, Waltham, MA). Primary antibodies included: rabbit anti-Tmem106b from Bethyl Laboratories (A303-439A), rabbit anti-Tmem106b generously shared and derived in the lab of Dr. Fenghua Hu, sheep anti-mouse progranulin (AF2557; R&D systems, Minneapolis, MN), goat anti-human progranulin (AF2420; R&D systems, Minneapolis, MN), mouse anti-Gapdh (H86504M; Meridian Life Sciences, Cincinnati, OH), mouse anti-Lamp1 (sc-20,011; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-C9ORF72 (ABN1645; Millipore), mouse anti-HA (clone12CA5; #11583816001; Roche, Indianapolis, IN), and goat anti-Cathepsin-D (clone C-20; sc-6486; Santa Cruz Biotechnology, Dallas, TX). Bands of Western blots were quantified using Image J (NIH, Bethesda, MD).

Cells were lysed in RIPA lysis buffer in the presence of protease-inhibitors (Roche). Following electrophoresis using NuPAGE 4–12% gels (Thermo Fisher Scientific), proteins were transferred onto 0.45 μm nitrocellulose membranes and the membranes were incubated overnight at 4°C with the indicated primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. The following antibodies were used in this study: mouse anti-LAMP1 (Santa Cruz, sc-20011), rabbit anti-LAMP2A (Abcam, ab18528), rabbit anti-actin (Sigma, A2066), mouse anti-Rab11 (Thermo Fisher Scientific, MA1-24919), rabbit anti-SQSTM1/p62 (Cell Signaling, 5114), rabbit anti-LC3B (Cell Signaling, 2775), and goat anti-Megalin (Santa Cruz, sc-16476).