Anti rat hrp

All blots were blocked with 10% skim milk powder in 0.1% PBS-Tween20. For the GST-pulldown, 1/1000 dilution of anti-HA rat antibody (abcam) was used followed by 1:2000 dilution of anti-rat HRP (GE Healthcare). To measure dE2F1a and dE2F1b transgene overexpression efficiencies, 10 L3 eye discs were dissected from control, GMRG4>FM-dE2F1a, and GMRG4>FM-dE2F1b overexpression groups. Anti-Myc (1/1000 dilution, DSHB) and Anti- β-tubulin (1/1000 dilution, DSHB) were used as primary antibodies followed by anti-mouse HRP (1/2000, GE Healthcare).

Primary mouse antibodies used for western blotting and immunostaining were rat anti-K8 and rat anti-K19 (Troma I respectively Troma III, Hybridoma bank, Iowa, USA), mouse anti-K7 (RCK 105, Abcam, Cambridge, UK), mouse anti-K20 (IT-Ks 20.10, Progen, Frankfurt, De), rat anti-K18 (Troma II, Hybridoma bank, Iowa, USA) [24 (link)], mouse anti-tubulin (Sigma, Munich, Germany), rabbit anti-caspase-7 and anti-cleaved caspase-7 (Cell Signaling, Danvers, MA, USA), rabbit anti-MPO (Thermo Scientific, Waltham, MA, USA) and rat anti-Hsc70 (Stressgen, Victoria, Canada). The secondary antibodies used for staining were Alexa 488 or Alexa 546 anti-mouse, Alexa 488 anti-rat and Alexa 488 anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA). The secondary antibodies used for western blotting were: anti-mouse HRP (GE Healthcare, Little Chalfont, UK), anti-rabbit HRP (Cell Signaling, Danvers, MA, USA) and anti-rat HRP (GE Healthcare, Little Chalfont, UK) antibodies. Nuclei were stained with Draq5 (Cell Signaling, Danvers, MA, USA) or Dapi (Invitrogen Carlsbad, CA, USA). Antibodies used for FACS analysis were anti-CD4-FITC and anti-CD49d-PE or anti-L-selectin-PE (Immunotools, Friesoythe, Germany).

Protein was isolated from parasites by resuspending harvested parasites in RIPA lysis buffer supplemented with a protease inhibitor cocktail (Research Products International Corp P506001). Samples were then sonicated using a QSonica Q800R3 at 50% amplitude for 2 min. Insoluble material was pelleted and removed. Protein concentrations of lysates were determined using a BCA protein assay kit (Thermo Fisher Scientific 23227), and 50 ug of protein was used for Western blotting. Protein samples were separated by SDS-PAGE in 4%–15% Bis-Tris gels with MOPS buffer and transferred to nitrocellulose membrane. Membranes were blocked in 5% non-fat milk and incubated in primary and secondary antibodies diluted in 5% non-fat milk. The following antibodies were used: anti-myc-HRP (Santa Cruz sc-40) diluted 1:100, anti-HA (Roche 27573500) diluted 1:2,000, anti-rat-HRP (GE NA935) diluted 1:2,000, anti-p30 (SAG1) (Invitrogen MA183499) diluted 1:2,000, and anti-mouse-HRP (GE NA931) diluted 1:2,000. Pierce Enhanced Chemiluminescence (ECL) detection reagent (Thermo Fisher Scientific 32109) and a BioRadV3 Chemidoc Imager were used to visualize blots.