Anti lamp1 d2d11

HEK 293T and C2C12 cells transfected with the respective expression constructs were washed in PBS and fixed using 4% paraformaldehyde for immunofluorescence test. Cells were blocked with 10% normal donkey serum and 0.3% Triton X‐100 in PBS for 60 min, incubated with primary antibody mentioned above (anti‐EGFP or anti‐LC3B) and anti‐LAMP1 (D2D11, 1:200, Cell Signaling Technology) in blocking solution at 4°C overnight and incubated with Alexa Fluor 488 or 594 secondary antibodies (1:1000, Life). DAPI (4′,6‐diami‐dino‐2‐phenylindole) (1:10,000, Life) was used for nucleic acid staining. Images were taken with a Zeiss 710 confocal microscope.

For standard immunofluorescence experiments, cells were grown on glass-bottomed six-well plates and fixed with 4% formaldehyde. Blocking was performed with 3% BSA in PBS with 0.1% Triton X-100. Primary and secondary antibody dilutions were performed in the same solution. We used anti–protein disulfide isomerase (2792; Abcam) to detect ER, anti-LC3 (2775S; Cell Signaling Technology) to detect autophagy, anti-LAMP1 (D2D11; Cell Signaling Technology) to detect lysosomes, anticatalase (D4P7B; Cell Signaling Technology) to detect peroxisomes, and anti-GM130 (D6B1; Cell Signaling Technology) to detect the Golgi apparatus. We used as a secondary antibody Alexa Fluor 568–conjugated secondary antibodies (Life Technologies). Nuclei were stained with 1 µg/ml Hoechst 33342 (Life Technologies) during one of the post–secondary antibody washes.