Rat monoclonal anti ha

Wild-type mouse ASIC2a constructs (untagged and N-terminal HA-tagged) have been described previously [13 (link), 48 (link)]. Truncations and point mutations in ASIC2a were generated with a Quickchange mutagenesis kit (Agilent Technologies). All constructs were verified by sequencing. The ASIC2 antibody was generated by immunizing rabbit with a C-terminal peptide corresponding to the last 20 amino acid of ASIC2a [17 (link)]. Other antibodies used were: mouse anti-tubulin (University of Iowa Developmental Hybridoma Bank), rat monoclonal anti-HA (Roche, Switzerland), mouse monoclonal anti-HA (Santa Cruz Biotech., Santa Cruz, CA and Syd Labs, Malden, MA), Dylight 680-, Dylight 800-, Alexa 680- and 800-conjugated secondary antibodies (Pierce, Rockford, IL; Invitrogen, CA; Li-cor, Lincoln, NE). Other reagents used: Endo H and PNGase F (New England Biolabs, Ipswich, MA); NHS-sulfo-LC-biotin and NeutrAvidin Beads (Pierce); culture media and serum (HyClone or Invitrogen); lipofectamine 2000 (Invitrogen).

HeLa cells (2 × 10⁶) were co-transfected with the expression constructs in 10 cm dishes with 2 μg each of His-SUMO1 and HA-tagged wild-type HMGXB4 or mutants. At 48 h post-transfection, cells were lysed by radioimmunoprecipitation assay (RIPA) buffer containing 10 mM N-ethylmaleimide (NEM) and protease inhibitors. For immunoprecipitation, equal amounts of lysates (containing 5 mg of total cellular protein from HeLa cells) were precleared with protein G-agarose beads (Sigma, St. Louis, MO, USA). Precleared extracts were incubated with 1 μg rat monoclonal anti-HA (Roche, Mannheim, Germany) for 2 h at 4 °C. Precipitates were washed extensively in lysis buffer; bound complexes were eluted with 2 × SDS–PAGE sample buffer and resolved by 7.5–10% SDS–PAGE. Immunoblotting was performed according to standard procedures, and proteins were detected with the anti-HA antibodies. Antibodies were detected by chemiluminescence using ECL Advance Western Blotting Detection Kit (GE Healthcare, Chicago, IL, USA).

Co-immunoprecipitations were performed 72 h after transfection. Cells were lysed in NP40 lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP40, pH 8.0), incubated for 30 min at 4 °C, and centrifuged for 30 min at 15.000 RPM. The protein concentration was estimated using a Bradford Protein Assay (BioRad). For co-immunoprecipitation experiments, 1 mg/mL total cell lysate was incubated overnight at 4 °C with 2 μg of “catching” antibody (mouse monoclonal anti-GFP (Abcam), rat monoclonal anti-HA (Roche), mouse monoclonal anti-mCherry, or rabbit polyclonal anti-Ca_v3.2 (Alomone). For co-immunoprecipitation of native proteins, 10% rat brain homogenate was prepared in lysis buffer (50 mM TrisHCl pH 8.0, 300 mM NaCl, 0.5% Triton ×100, 0.5% DOC, Protease Inhibitors without EDTA) and stored as aliquots of 500 ml at −80 °C. For Co-immunoprecipitation, 500 μL of lysis buffer without detergent and 2 μg of “catching” antibody (rabbit polyclonal anti-CNX (Abcam) or rabbit polyclonal anti-Ca_v3.2 (Santa Cruz)) were added to the 10% brain homogenate samples and incubated overnight at 4 °C. Samples were then incubated with 30 μL (50% slurry) Sepharose G beads for 2 h at 4 °C. Beads were centrifuged for 1 min at low speed, washed three times (20 mM Tris, 300 mM NaCl, 0.1% Tween-20, pH 8.0), and incubated with 30 μL Laemmli buffer.

Rat polyclonal anti-PHY2 antibody was raised against the GST-tagged PHY2 (aa 231-297) that was bacterially expressed, and were affinity-purified against the MBP-tagged antigenic proteins that were covalently coupled to CNBr-activated Sepharose 4B.
Rat anti-PHY2 (1 ng/μl), rabbit anti-actin (1:800 dilution; Cat No: A5060; Sigma-Aldrich) mouse monoclonal anti-FLAG (1:1,000 dilution; Cat No: F1804; Sigma-Aldrich) and rat monoclonal anti-HA (1:1,000 dilution; Cat No: 1867423; Roche) were used for Western blotting.
The following primary antibodies were used for immunohistochemistry: mouse monoclonal anti-calbindin (1:500 dilution; Cat No: 214011; Synaptic Systems, Goettingen, Germany), mouse monoclonal anti-vGluT2 (1:300 dilution; Cat No: MAB5504; Millipore, Billerica, MA, USA), and rabbit anti-VGAT (1:500 dilution; Cat No: AB5062P; Millipore) antibodies.

HepG2 cells were infected with WT-F or ZIPCO-HA sporozoites, and samples were fixed with 4% PFA at 48 h postinfection. After incubation with rat monoclonal anti-HA (Roche) and a rabbit anti P. berghei MSP1 polyclonal serum (de Koning-Ward et al, 2003 (link)) followed by Alexa Fluor 568 and 647 conjugated antibodies, samples were stained with DAPI and mounted in ProLong® Antifade kit (Molecular probes). 16-bit images were acquired at the Imaging Platform of the Pasteur Institute (PFID) on a laser scanning confocal microscope (LSM 700 Zeiss) with a 63× Plan-APOCHROMAT/oil objective (Zeiss). Images were processed using the ImageJ software (Schneider et al, 2012 (link)).

Freshly lysed parasites were collected by centrifugation at 1,000 g for 10 minutes. These samples were suspended in PBS containing 1% Triton X-100, or PBS with 10 mM deoxycholate for 15 minutes at room temperature. For immunoblot analysis, samples were centrifuged at 12,000 g for 30 minutes to separate the pellet from the soluble fraction. The latter was acetone-precipitated and both fractions were resuspended in the same volume of loading buffer before being separated by SDS-PAGE and analysed by immunoblot. The primary antibodies used for detection and their respective dilutions were: mouse monoclonal anti-α-tubulin (clone B-5-1-2, Sigma-Aldrich) at 1/4000, rabbit polyclonal anti-TgMORN1 at 1/100012 (link), rabbit polyclonal anti-TgIF2α at 1/50045 (link) and rat monoclonal anti-HA at 1/1000 (clone 3F10, Roche).
Detergent-extracted parasite samples for immunofluorescence were settled onto poly-L-lysine-coated glass slides (Thermo Scientific) for 15 minutes at room temperature. These samples were fixed with 4% (w/v) paraformaldehyde in PBS, prior to processing for immunofluorescence (see below).

Protein extracts were fractioned in SDS-PAGE and transferred to a nitrocellulose membrane. Transferred proteins were visualized with Ponceau S staining. Membranes were treated with 10% non-fat milk in PBS for 2 h and then incubated with specific antibodies diluted in 0.5% Tween 20 in PBS (PBS-T) for 3 h. Primary antibodies used were: rat monoclonal anti-HA 1:2000 (ROCHE), affinity-purified rabbit polyclonal anti-TcATAT 1:200, mouse monoclonal anti-trypanosome α-tubulin clone TAT-1 1:1000 (a gift from K. Gull, University of Oxford, UK), rabbit polyclonal anti-Acetyl-lysine 1:1000 (Millipore), mouse monoclonal anti-acetylated α-tubulin clone 6-11B-1 1:2000 (Sigma Aldrich). Bound antibodies were detected using peroxidase-labeled anti-rabbit IgG (GE Healthcare), anti-mouse IgG (GE Healthcare) or anti-rat IgG (Thermo Scientific) and developed using ECL Plus kit (GE Healthcare) according to manufacturer’s protocols. Immunoreactive bands were visualized and photographed in the Amersham Imager 600 (GE Healthcare). Images were processed and bands where quantified with ImageJ (Schindelin et al., 2012 (link)).