Imagequant 800 western blot imaging system

Total proteins were extracted from 1.0 × 10⁸ parasites grown on media supplemented with 0.5 mM IAA at different times or as indicated in the figure legends. Total proteins were extracted by resuspending the cell pellet in Laemmli buffer and boiling for 5 min. Protein samples were run on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane overnight. Primary and secondary antibody incubations were done in 5% milk–PBS with rabbit anti-HA at 1:500 (Cell Signaling, catalog no. 3724S) and anti-rabbit HRP at 1:10,000 (Bio-Rad, catalog no. 1721017). Images were obtained using an ImageQuant 800 Western blot imaging system (Amersham) with exposure for a total of 30 s.

Dishes with myotubes were washed 3x with cold PBS on ice. Cells were removed from ice and 200 μl of lysate buffer (1% SDS in 100 mM TRIS–HCl, pH 8) was added to the dishes. Cells were scrapped and transferred into 1.5 ml Eppendorf. To remove matrigel, cells were spun down for 5 min at 240g. The supernatant was collected and stored at −80°C. For Western blots, cell lysate protein concentrations were measured using the BCA kit (Pierce). Samples were mixed with 1:4 lammeli buffer and boiled at 95°C for 5 min. The same amount of the sample (10 or 30 ng/ml) was loaded onto 4–15% pre-cast Bis–Tris gel (Invitrogen) and run at 100 V. Proteins were transferred onto nitrocellulose membrane for 75 min at 100 V. Subsequently, membranes were blocked for 1 h in blocking buffer (BB) using 5% non-fast dry milk in TBS-T (TRIS-buffered saline with 0.1% Tween 20). Primary antibodies in BB were incubated O/N at 4°C. Membranes were washed 3x with TBS-T under agitation and then incubated with HRP-conjugated secondary antibodies for 1 h at RT. Membranes were washed 3x in TBS-T under agitation, visualized using ECL reagent (Peirce), and imaged using Amersham ImageQuant 800 Western blot imaging system. Quantification was done in Image Laboratory. All used antibodies are listed in Table S1.