Enhanced chemiluminescence detection

Cells were lysed in RIPA buffer for 30 min on ice. Protein lysates (25 μg) were loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Gels were transferred to nitrocellulose membrane and blocked in 5% non-fat milk in Tris buffer, pH 8.0, at room temperature for 2 h. The membrane was blotted with anti-TTP Primary Ab (clone 3A2; 1 mg/mL; Santa Cruz Biotechnology) in Tris buffer containing 5% milk powder and left overnight at 4°C. After extensive washing, blots were subjected to horseradish peroxidase–conjugated sheep anti-mouse Ig secondary Ab at a 1:5,000 dilution in 5% milk and then detected with enhanced chemiluminescence detection (PerkinElmer Life Sciences Inc., Boston, MA, United States).

Purified parasites were washed in PBS and collected by centrifugation. Total lysates were obtained by resuspending the parasite pellets with Leammli loading dye, heated at 95°C for 10 min, and briefly sonicated. After separation on the SDS-PAGE gels, proteins were transferred onto nitrocellulose membrane and probed with monoclonal antibodies against HA- (rat 3F10, Roche Applied Sciences), myc-epitope (mouse, Cell Signaling Technology), and α-Tubulin (mouse 12G10, kindly provided by Dr. Jacek Gaertig, University of Georgia, GA, US). After incubation with secondary HRP-conjugated anti-mouse or anti-rat antibodies, proteins were visualized by enhanced chemiluminescence detection (PerkinElmer).

Filter-purified parasites were washed in PBS and collected by centrifugation. Total lysates were obtained by resuspending the parasite pellets in the Leammli loading dye, heated at 95°C for 10 min, and briefly sonicated. After separation on the SDS-PAGE gels, proteins were transferred onto nitrocellulose membrane and probed with monoclonal α-HA (rat 3F10, Roche Applied Sciences), α-myc (mouse, Cell Signaling Technology), and α-Tubulin A (mouse 12G10, kindly provided by Jacek Gaertig, University of Georgia) antibodies. After incubation with secondary HRP-conjugated anti-mouse or anti-rat antibodies, proteins were visualized by enhanced chemiluminescence detection (PerkinElmer).

For each protein sample 10 ug was loaded on SDS PAGE gel (1.5 mm). Migration of proteins in the PAGE gel was conducted at 150 V until the blue band from the sample buffer run out of the gel. Protein-Marker IV was also loaded to determine the molecular weight of the proteins, Proteins were then transferred onto a Polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The following antibodies were used in the western blot analysis: actin (ACT), ADP-glucose pyrophosphorylase (ADGP), β-amylase, isocitrate lyase (ICL), aquaporin, plasma membrane intrinistic protein 1–3 (PIP1), tonoplast intrinsic protein 1-1(TIP1), heat shock protein 90 (HSP90), dehydroascorbate reductase (DHAR1), and alpha-amylase (from Agrisera, Sweden). The PVDF membrane was probed with primary antibody and developed using enhanced chemilu-minescence detection (PerkinElmer, Waltham MA, USA). The blots were detected using the BeyoECL plus (P0018). The images were obtained with the ChemiDoc ^TM MP imaging system, and the quantifications were conducted with the software Image Lab ^TM V5.1.

Cells were washed with phosphate buffered saline (PBS), lysed and centrifuged at 14.000 xg for 10 min at 4°C. For EGFR detection, lysis buffer was 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 0,5% Nonidet P-40 (Garcion et al., 2004 (link)). For detection of all other proteins, we used RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40 and 1% sodium deoxycholate). Both buffers were supplemented with protease and phosphatase inhibitors mixture (Roche Applied Science, Mannheim, Germany). Proteins were resolved by SDS-PAGE and transferred into PVDF membranes. Blots were subsequently incubated with antibodies as follows: Anti-PPARβ/δ (1:1000), anti-PPARγ (1:1000), anti-EGFR (1:1000), anti-Nestin (1:5000), anti SOX2 (1:2000). For detection, horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were used followed by enhanced chemiluminescence detection (Perkin Elmer Inc., Waltham, MA). Optical density was quantified with ImageJ software.

Parasites were inoculated onto confluent HFF coverslips and incubated for the times indicated, and IFA was performed with the following primary reagents: anti-HA antibody (Roche rat monoclonal antibody [MAb] 3F10, 1:500), anti-myc antibody (Santa Cruz Biotechnology mouse MAb, 1:500), biotin-labeled DBA (1:3,000; Vector Labs, CA), and 4',6-diamidino-2-phenylindole (DAPI; 0.5 mg/ml). All Alexa (Molecular Probes, CA)- and streptavidin (Vector Labs, CA)-conjugated secondary antibodies were used at 1:1,000. Image acquisition was performed with a Zeiss Axiovert microscope equipped with a 100× objective. Western blotting with a specific antibody monitored the protein expression in Toxoplasma parasites. Purified parasites were lysed in SDS-PAGE sample buffer with Laemmli loading dye, heated at 95°C for 10 min, and briefly sonicated. After separation on the gel, proteins were transferred onto nitrocellulose membrane, detected with MAbs and horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, PA), and visualized by enhanced-chemiluminescence detection (PerkinElmer).