Anti β tubulin

The expression of PfDOZI::GFP and its subcellular localization were directly visualized using the GFP signal under the Olympus FV1000 confocal microscope using a 60× oil objective and processed by FV10-ASW imaging software. Nuclei were counter-stained with DAPI. For IFA, parasite cultures were coated on slides, allowed to air-dry, and then fixed by methanol: acetone (1:1) for 20 min at −20 °C. The samples were treated with 0.01% Triton X-100 for 5 min to permeabilize the parasite cell membrane and 5% BSA in PBS for 30 min at room temperature to block non-specific binding sites. Subsequently, the slides were incubated with primary antibodies in 3% BSA/PBS at 4 °C overnight and then secondary antibodies at room temperature for 1 h. Depending on the purpose, the primary antibodies included anti-PABP1 (1:1000)^{32 (link)}, anti-Alba4 (1:1000), anti-α-tubulin II (1:2000) for male gametocytes, anti-GFP (1:500, Invitrogen), anti-β-tubulin (1:250, Sigma), anti-PfAMA1 (1:250, MR4), and anti-PfMSP5 (1:500, MR4). Secondary antibodies were Alexa Fluor 488 or 594 conjugated goat anti-mouse or donkey anti-rabbit IgG antibodies. The slides were mounted with Vectashield antifade mounting medium with DAPI and observed under an Olympus FV1000 confocal microscope. ImageJ software was used to measure the fluorescence density.

Proteins were isolated from cells and tissues using the following methods. Cells from cell culture were washed with phosphate-buffered saline and collected in lysis buffer and processed as previously outlined^{87 (link)}. Protein content was determined using the BioRad protein assay. Equal amounts of whole protein lysate were separated on SDS-polyacrylamide gels and transferred to activated polyvinylidene fluoride membranes (Millipore) Western blotting was done in keeping with standard protocols. Primary antibodies used were: anti-HELLS (ABD41, Millipore), anti-Cyclin D2 (sc-593, Santa Cruz), anti-Cyclin D1 (NBP2-32840, Novus Biologicals), anti-β-actin (4970S, Cell Signaling Technology), anti-β-tubulin (T4026, Sigma), and anti-cleaved caspase-3 (9661L, Cell Signaling Technology). Secondary antibodies conjugated to horseradish-peroxidase were: anti-mouse (715-035-150, Jackson Immuno Research), and anti-rabbit (31460, Pierce, Life Technologies). Blots were developed using Pierce ECL reagents, and chemiluminescence was detected by exposing membranes to GE-Amersham film. Blots were scanned into digital files and exposures were selected for densitometry. Densitometric analysis was done using ImageJ.

Western blotting was performed as previously described (Abdelhamed et al., 2018 (link); Emmert et al., 2019 (link)). Briefly, whole P11 rat brain lysates in RIPA buffer [50 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 1% proteinase inhibitor cocktail (Thermo Fisher Scientific)] were separated, transferred to a PVDF membrane and probed with anti-CCDC39 (1:1000, #HPA035364, Sigma-Aldrich), anti-β-tubulin (1/1000, #T8660, Sigma-Aldrich), and anti-rabbit/mouse IgG-IRDye680RD/800CW (LI-COR) antibodies. Fluorescent signals were detected using the Odyssey Imaging System (LI-COR).

Western blot analysis was performed on retinae, which were harvested. Samples were lysed in hypotonic buffer (10 mM Tris-HCl [pH 7.5], 10 mM NaCl, 1,5 mM MgCl₂, 1% CHAPS, 1 mM PMSF, and protease inhibitors) and 20 µg of these lysates were separated by 12% SDS-PAGE. After the blots were obtained, specific proteins were labeled with anti-1D4 antibody anti-Rhodopsin-1D4 (1:1000; Abcam, Cambridge, MA), and anti-β-tubulin (1:10000; Sigma-Aldrich, Milan, Italy) antibodies.