Goat anti rat hrp

Late trophozoite parasites cultured with or without aTc were isolated using a 65% Percoll gradient. Recovered parasites were resuspended in Laemmli Sample Buffer (Bio-rad) and β-mercaptoethanol (Sigma-Aldrich) and loaded on a 10% Mini-PROTEAN® TGX™ Precast Protein Gel. Samples were transferred to 0.2 μm polyvinylidene difluoride (PVDF) membrane using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked in 5% skim milk in 0.1% PBS-Tween (PBS-T) for 1 h at room temperature and incubated overnight with rat anti-HA tag (Roche) and mouse anti-Actin (Invitrogen) at 1:3000 in 2% Bovine Serum Albumin (BSA). The blot was then washed 3 times in PBS-T and probed with goat anti-rat HRP (Biolegend) and goat anti-mouse HRP (Biolegend) at 1:10,000 in 2% BSA PBS-T for 1 h at room temperature. The blot was imaged on ChemiDoc MP (Bio-Rad) in Clarity Max Western ECL Substrate (Bio-Rad). Western blot analysis was done using Image Lab v6.0 (Bio-Rad).

Cells were lysed on ice with lysis buffer containing 20 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and protease inhibitors (Millipore). 0.5 × 10⁶ cells were lysed for each condition and run on SDS-PAGE gels, transferred to nitrocellulose (Bio-Rad), and blotted using standard procedures. Rat anti-mouse Bim mAb (3C5) was purchased from Enzo Life Sciences. Goat anti-rat HRP was purchased from BioLegend. Rabbit anti-mouse actin mAb (D18C11) was purchased from Cell Signaling. Goat anti-rabbit HRP was purchased from Bio-Rad.

Samples from individual mice were separated using SDS-PAGE under reducing conditions for mC3 and non-reducing conditions for mC5, hFH and mFH. Goat anti-mC3 (MP Biomedicals), goat anti-hC5 (Quidel), rat anti-mFH (R&D Systems) or goat anti-hFH (Quidel) were used as detection antibodies followed by goat anti-rat-HRP (Biolegend), mouse anti-goat/sheep IgG-HRP (Sigma) and rabbit anti-mouse IgG-HRP (Dako), as required. Blots were visualized using ECL Western Blotting Substrate (Pierce, Thermo).