Odyssey block buffer

For width measurements, we incubated C2C12 myotubes with nucleic acid stain, Syto13 (2.5 μM, Invitrogen) for 15 min. We captured live-cell bright field and fluorescence images (excitation: 488 nm, emission 509 nm) using a CCD camera (CoolSNAP-ES, Roper Scientific Photometrics) attached to a Nikon TE2000 microscope with NIS Elements image acquisition software (Nikon). To obtain the average myotube width per well, we measured 3 images per well, 5 myotubes per image, and 5 width measurements per myotube. For CD13 immunofluorescence, myotubes were fixed in 100% methanol cooled to −20C for 5 min, after which they were blocked for 30 min with Odyssey block buffer (LI-COR). After blocking, we incubated cells with 1:250 dilution of rabbit anti-CD13 antibody (Abcam) overnight at 4C. After rinsing myotubes 3x, we incubated cells 1 h with 1:1000 secondary antibodies, donkey anti-rabbit 649 (DyLight). We captured fluorescence images using the same system as above (excitation: 655 nm, emission 670 nm). Fluorescence was measured with ImageJ (NIH).

Fluorescence microscopy was performed on live, GFP-expressing parasites using a Zeiss Axioskope. Nucleic acid was detected by staining with DAPI. For western blotting, 3 × 10⁸ parasites were released from iRBCs with 0.035% saponin and lysed in Ripa buffer. For each sample, 9 × 10⁷ parasites were loaded onto a 12% TGX gel (BioRad). Proteins were transferred onto PVDF using wet transfer with 20% methanol. Blots were blocked overnight at 4 °C with Licor Odyssey block buffer. Primary antibodies were LivingColors mouse-α-GFP (Takara, Cat. No. 632380) (1:500) and rabbit-α-HAD1 (1:1,000) (a gift from Dr Audrey R. Odom, Washington University25 (link)). Secondary antibodies were goat-α-mouse (800) and donkey-α-rabbit (680) IR-Dyes (1:20,000) from Licor.

Ten to 20 μg of proteins from lysosomal fraction were separated by 4–12% NuPAGE Bis-Tris gel and transferred to nitrocellulose membrane using iBlot (Invitrogen, P3 7min). Blots were blocked with Odyssey Block Buffer (Licor) for 1 h and probed overnight with primary antibodies (TMEM175 1:500, Proteintech Group 19 925-1-AP; MycTag [AAV TMEM175] 1:1000, Cell signaling 2276; LAMP1 1:500 Abcam, ab24170; GAPDH 1:1000, Abcam, ab9485). Appropriate IRDye-conjugated secondary antibodies (1:10000, Licor) were used to visualize proteins with Odyssey imaging system (Licor).