A1122

The purified SAS4-GFP gametocytes were activated in ookinete medium then fixed at 2–8 min post-activation with 4% PFA diluted in microtubule stabilizing buffer (MTSB) for 10–15 min and added to poly-L-lysine coated eight-well slides. Immunocytochemistry was performed using primary GFP-specific rabbit monoclonal antibody (mAb) (A1122; used at 1:250; Invitrogen) and primary mouse anti–α tubulin mAb (T9026; used at 1:1,000; Sigma-Aldrich). Secondary antibodies were Alexa 488–conjugated anti-mouse IgG (A11004; Invitrogen) and Alexa 568–conjugated anti-rabbit IgG (A11034; Invitrogen) (used at 1 in 1,000). The slides were then mounted in VECTASHIELD 19 with DAPI (Vector Labs) for fluorescence microscopy.

The purified gametocytes from kinesin-8B–GFP, WT-GFP, and Δkinesin-8B parasites were activated in the ookinete medium and then fixed at 0 min, 1–2 min, 6–8 min, and 15 min postactivation with 4% PFA (Sigma-Aldrich) diluted in microtubule stabilising buffer for 10–15 min and added to poly-L-lysine–coated slides. Immunocytochemistry was performed using primary GFP-specific rabbit monoclonal antibody (mAb) (A1122; Invitrogen; used at 1:250) and primary mouse anti-α tubulin mAb (T9026; Sigma; used at 1:1,000). Secondary antibodies were Alexa 488–conjugated antimouse IgG (A11004; Invitrogen) and Alexa 568–conjugated antirabbit IgG (A11034; Invitrogen) (used at 1 in 1,000). The slides were then mounted in Vectashield 19 with DAPI (Vector Labs) for fluorescence microscopy. Parasites were visualised on a Zeiss Axio Imager M2 microscope fitted with an AxioCam ICc1 digital camera (Carl Zeiss, Inc).
To measure nuclear DNA content of activated microgametocytes by direct immunofluorescence, images of parasites fixed (0 mpa and 8–10 mpa) and stained as above were analysed using the ImageJ software (version 1.44) (National Institute of Health) as previously described (Tewari et al, 2005 (link)).

To determine the in vivo DNA-binding activity of CjWRKY1 using a ChIP assay, approximately 4 × 10⁶ protoplasts isolated from 2- to 3-week-old 156-S cultured cells were transfected with 300 μg of 35S::sGFP (as vector control) or 35S::CjWRKY1-sGFP plasmid vector and incubated for 24 h at 24°C in the dark. Then, protoplasts were suspended in 1 ml of W5 solution (154 mM NaCl, 125 mM CaCl₂⋅2H₂O, 5 mM KCl, 5 mM glucose, pH 5.8) containing 1% formaldehyde for 10 min to allow cross-linking. Then, immunoprecipitation of chromatin was performed as previously described (Yamada et al., 2011a (link)) with Dynabeads Protein G (Invitrogen, Oslo, Norway) and 5 μl of anti-GFP antibodies (A1122, Invitrogen, Carlsbad, CA, USA). The purified chromatin was subjected to PCR for 33 cycles with specific primer pairs for the detection of genomic regions corresponding to the promoter and the control.