G1546

To detect mitochondrial GFP in RH pSAG1-βGal/pmt-GFP parasites, samples taken during organelle enrichment were directly diluted in Laemmli buffer (0.2 M Tris–HCl, 8% (w/v) SDS, 40% glycerol, 20% (v/v) β-mercaptoethanol, 0.005% bromophenol blue), incubated at 95 °C for 5 min and subjected to sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). SDS‒PAGE and immunoblotting were carried out as described previously (Kupsch et al., 2012 (link)). For immunodetection, monoclonal primary mouse anti-GFP antibody (G1546, Sigma‒Aldrich) and polyclonal rabbit anti-heat shock protein 70 antibody (AS05 083 A, Agrisera) followed by secondary horseradish peroxidase (HRP)labeled goat antimouse antibody (ab205719, Abcam) and goat anti-rabbit antibody (ab205718, Abcam) were used.
Proteins from sucrose gradient fractions were isolated using methanol-chloroform-water extraction (Wessel and Flügge, 1984 (link)). Protein pellets were resuspended in Laemmli buffer, heated for 5 min at 95 °C, and subjected to SDS-PAGE and immunoblotting. Primary rat anti-HA antibody (11867423001, Roche) and secondary rabbit anti-rat (HRP) antibody (ab6734, Abcam) were used for TguL11m:HA detection.

Larvae were fixed with 2% paraformaldehyde in PBS for 25 min to overnight depending on the stage. Immunodetection for slow myosin heavy chain (MyHC) (1:5, F59; Devoto et al., 1996 (link)), general MyHC (1:10, A4.1025; Blagden et al., 1997 (link)), Pax7 (1:5; DSHB; Kawakami et al., 1997 (link)), Myogenin (1:50, sc-576, Santa Cruz) and GFP (1:500, TP-401, Torrey Pines or 1:500, G1546, Sigma) was performed in PBS with 0.5-1% Triton X-100 (PBT) for between overnight and 5 days at 4°C on a rotary shaker, depending on larval age and antigen (Hinits and Hughes, 2007 (link)) followed by Alexa Fluor 488 or 555 secondary antibodies (1:1000; A21121 and A21428, respectively; Invitrogen). at least overnight (±Hoechst 33342) at 4°C. After EdU detection in Fig. S4, samples were blocked in 5% BSA, 3% normal goat serum, PBT for 20 min, incubated using Alexa Fluor 488-conjugated anti-GFP (1:500, A-21311, Molecular Probes) and Hoechst in block buffer (shaking at 4°C for 3-6 h), followed by 6×15 min washes in PBT. Phalloidin-Alexa Fluor 555 or phalloidin-Alexa Fluor 633 (1:1000, A34055 or A22284, Thermo Fisher) were used to-stain F-actin. Larvae were mounted on glass slides under bridged coverslips in Citifluor AF1 or Vectashield (H-1000, Vector Laboratories). In situ mRNA hybridisation was as described (Groves et al., 2005 (link)).