Rabbit monoclonal anti myc

Parasites from 9 mL of P. falciparum culture were isolated by saponin lysis, washed with PBS and resuspended in 1 x NuPAGE LDS sample buffer (Invitrogen). Proteins were separated by electrophoresis on 4–12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. After blocking, membranes were probed with 1:2000 monoclonal mouse anti-FLAG M2 (Sigma) and 1:10,000 IRDye 680RD goat anti-mouse IgG (LiCor Bioscience, Lincoln, NE) for anti-FtsH1 immunoblots. For anti-PDF immunoblots, membranes were probed with 1:2000 rabbit monoclonal anti-MYC (Cell Signaling Technology 2278S, Danvers, MA), followed by 1:20,000 rabbit polyclonal anti-PfAldolase (Abcam ab207494, UK) and 1:10,000 donkey anti-rabbit 800 (LiCor Biosciences). Fluorescence antibody-bound proteins were detected with Odyssey Imager (LiCor Biosciences). When antibodies of the same species were used, membranes were probed and imaged sequentially. Immunoblots of FtsH-FLAG and PFD-myc were repeated in the laboratory >2 times.

The ChIP assay 19 (link) was performed using a SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, USA) according to the manufacturer's protocols. Specifically, approximately 1.2×10⁷ SiHa cells were cross-linked in 1% formaldehyde; after 10 min, glycine was added to terminate the reaction. Adherent cells were scraped off in PBS containing a protease inhibitor cocktail and centrifuged at 2000 × g for 5 min to collect the precipitate. Micrococcal nuclease was added to digest the DNA to the optimal length and 0.5 M EDTA was used to stop the digest. The nuclear membrane was then disrupted by ultrasound and the supernatant containing the cross-linked chromatin sample was collected. Rabbit monoclonal anti-MYC (Cell Signaling Technology) antibody, IgG antibody, and samples were incubated overnight at 4 °C for subsequent immunoprecipitation. ChIP-grade protein G magnetic beads were added to each IP reaction followed by shaking for 2 h. Then, the beads were cleaned with high/low salt solutions, the chromatin was eluted, and cross-linking was reversed. DNA was purified using spin columns and subjected to qRT-PCR. The primers used for ChIP-qPCR are listed in Table S2.

Samples were fixed at different time points in fresh 4% paraformaldehyde (PFA). The suspension was allowed to adhere onto poly-l-lysine coated slides overnight at 4°C. The slides were washed once with PBS and immunocytochemistry performed per manufacturer's instructions for rabbit monoclonal anti-myc 1:200 (Cell Signaling). Mouse monoclonal anti-alpha tubulin 1:500 (Sigma) was used simultaneously or independently using the same protocol. Secondary antibodies Alexa 488-conjugated anti-rabbit IgG and Alexa 568 conjugated anti-mouse IgG for fluorescence detection were used at 1:1000 (Molecular probes). The slides were mounted in Vectashield with DAPI (Vector Labs). Parasites were visualized on a Leica SP5 confocal microscope and acquired and analysed with the LAS AF Lite software (Leica). For quantification, samples were visualized on a Leica DMR microscope and imaged with the Zeiss AxioCam HRC and Axiovision software respectively. Cell nuclei diameters were measured using ImageJ and statistical analysis performed using Student's t-test.