Mouse anti dstyk

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).

Zebrafish embryos at 48 hpf and cultured COS-7 cells were lysed using RIPA lysis buffer containing protease inhibitors (Roche). Equal amount of protein samples was resolved on a 10% or 12% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore). Then samples were probed with the primary antibodies, including rabbit anti-S6K (Cell Signaling Technology, 2708, 1:1000), rabbit anti-p-S6K (T389) (Cell Signaling Technology, 9205, 1:1000), mouse anti-DSTYK (Santa Cruz, sc-374487, 1:500), mouse anti-LAMP1 (Santa Cruz, sc-20011, 1:500), rabbit anti-TFEB (Proteintech, 13372-1-AP, 1:1000), rabbit anti-LAMP3 (Proteintech, 12632-1-AP, 1:1000), mouse anti-actin (Sigma, A5316, 1:5000), overnight at 4 °C. Secondary antibodies goat anti-rabbit IgG (ZSGB-BIO, ZB-2301, 1:5000), goat anti-mouse IgG (ZSGB-BIO, ZB-2305, 1:5000) was used, as a secondary antibody, followed by detection with Pierce™ ECL Western Blotting Substrate (Thermo Scientific, 32106). Uncropped images of western blotting and gel are shown in the Source Data file.