Mouse monoclonal anti myc antibody

The antibodies used in the present project are as follows. Primary antibodies: rabbit polyclonal anti-tyrosine hydroxylase antibodies AB152 (Millipore, MA, USA); mouse monoclonal anti-Myc antibody (Cell Signaling Technology, Beverly, MA, USA); mouse monoclonal anti-actin antibody (Sigma-Aldrich, Rehovot, Israel); rabbit polyclonal anti-ERK antibodies (Santa Cruz Biotechnology, CA, USA). Secondary antibodies: horseradish peroxidase-conjugated goat anti-mouse antibodies; horseradish peroxidase-conjugated goat anti-rabbit antibodies, all from Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Primary antibodies used were as follows: Mouse monoclonal anti-myc antibody [1:1000 for western blotting (WB), 1:600 for immunoprecipitation (IP), 1:200 for immunofluorescence (IF), Cell Signaling Technology, Inc. Denver, MA, USA, #2276]; Polyclonal rabbit anti-GFP antibodies (1:1000 for WB, Santa Cruz Biotechnology, Dallas, TX, USA, #sc-8334); Polyclonal rabbit anti-Rab11 (1:30 for IF, Invitrogen, Camarillo, CA, USA, #71–5300); Monoclonal mouse anti-EEA1 antibody (1:30 for IF, BD Biosciences, San-Jose, CA, USA, #610456); Monoclonal Mouse anti-HA antibody (1:1000 for WB, Santa Cruz Biotechnology, Denver, TX, USA, #sc-805).
Secondary antibodies included: Peroxide-conjugated goat anti-mouse (1:5000 for WB, #115-035-003); Peroxide-conjugated goat anti-rabbit (1:10,000 for WB, #111-035-144); Cy3-conjugated goat anti-mouse (1:200 for IF, #115-166-072); Rhodamine Red-conjugated goat anti-rabbit (1:200 for IF, #111-295-144). All secondary antibodies were from Jackson Immunoresearch Laboratories, West Grove, PA, USA).

The following primary antibodies were used in this study: mouse monoclonal anti-myc antibody (1:1000 for Western blotting (WB); Cell Signaling Technology, Inc., Denver, MA, USA); mouse monoclonal anti-actin antibody (1:1000 for WB, Sigma-Aldrich, Rehovot, Israel); rabbit polyclonal anti-Erk antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and rabbit polyclonal anti-GlcCer antibodies (1:50 for confocal imaging; Glycobiotech, Kukels, Germany). Secondary antibodies used were: Alexa fluor 633 conjugated goat anti-rabbit antibodies (1:250 for confocal imaging; Invitrogen, Eugene, OR, USA); horseradish peroxidase-conjugated goat anti-mouse antibodies (1:5000 for WB, Jackson ImmunoResearch Laboratories, West Grove, PA, USA); and horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:10,000 for WB, Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

For expression analyses, proteins were extracted from yeast by alkaline lysis method as described [45 (link)]. Briefly, yeast cells were grown on liquid medium to an OD₆₀₀ of 1–2. After harvesting cells by centrifugation, the cells were resuspended in 200 μL of lysis solution (1.85 M NaOH, 7.4% β-mercaptoethanol) and incubated on ice for 10 min. Protein was precipitated by addition of 200 μL ice cold 50% trichloroacetic acid (TCA), pelleted by centrifugation, washed twice with 1 mL ice cold acetone, dried and resuspended in appropriate amount of 2x protein gel loading buffer (125 mM Tris pH 6.8, 4.0% SDS, 20% glycerol, 4.0% β-mercaptoethanol, 1 M urea, 0.05% bromophenol blue, 0.05% xylene cyanol). Western blot analyses were performed using anti-Myc mouse monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) for Myc-epitope tagged Gal4_BD-fusions from pGBKT7-based plasmids.
For expression of FLAG epitope-tagged ADA2, ADA3, GCN5 and AATF; CFP epitope-tagged ADA3, ADA2 and GCN5; YFP epitope-tagged AATF proteins in mammalian cells, the fusion proteins extracted from the cells and were subjected to western blot analysis using an anti-FLAG M2 (Sigma-Aldrich, Milwaukee, WI, USA) and anti-GFP antibodies (Proteintech Group, Rosemont, IL, USA), respectively.

Protein extracts were prepared by breaking the cells with glass beads and acid precipitation essentially as previously described 39 (link). Aceton washed protein pellets were dried, re-suspended in 1X Laemmli-sample buffer (0.2 M Tris-HCl [pH 6.8], 1.5% sodium dodecyl sulfate [SDS], 10% glycerol, 1 mM EDTA, 0.004% bromophenol blue) containing 50mM DTT, heated for 10 min at 65°C and separated by SDS-PAGE. Protein transfer and detection was done essentially as previously described 40 (link). The primary antibodies used included anti-HA rat monoclonal peroxidase-conjugated antibody (3F10; Roche), anti-Myc mouse monoclonal antibody (Cell Signaling CST2276), anti-myc-HRP (Thermo Fisher R951-25), anti-V5-HRP (Thermo Fisher R961-25), anti-Flag monoclonal antibody (M2 Sigma) and anti-Hexokinase polyclonal antibody.

Thin blood smears from cultured parasites were prepared, air-dried and fixed in a 1:1 acetone:methanol mixture at − 30 °C for 5 min^{31 (link)}. Smears were blocked with PBS containing 10% normal goat serum (Invitrogen) at 37 °C for 30 min and immunostained with mouse anti-myc monoclonal antibody (9B11, Cell Signaling Technology) at 1:500 dilution in PBS supplemented with 0.05% Tween-20 and incubated at 4 °C overnight. Double immunostaining of smears was done with rabbit anti-SBP4 at 1:1000 dilution. The smears were incubated with Alexa Fluor 488-conjugated goat anti-mouse or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (1:500; Invitrogen) at 37 °C for 30 min. Nuclei were stained by incubation of smears with 1 μg/mL Hoechst 33342 solution. The smears were examined using a confocal laser-scanning microscope (CS-SP5, Leica Micro-system, Wetzlar, Germany).

HEK293T cells were cultured on cover slips in 12-well plate at 80% confluence prior for transfection. 24 h after transfection with the WT or mutant SOX11 constructs, cells were washed by 1 x PBS and fixed using 4% paraformaldehyde for 15 min at room temperature. Samples were then washed with 1 × PBS three times and blocked in the blocking buffer (1x PBS/5% goat serum/0.3% Triton X-100) for 1 h. Coverslips were incubated with mouse anti-Myc monoclonal antibody (Cell Signaling Technology, Danvers, MA, United States) at 4°C overnight. The cover slips were then mounted on microscope slides using ProLong^® Gold Antifade reagent with DAPI (Cell Signaling Technology) and analyzed using a Leica DM6000 fluorescence microscope (Leica Microsystems, Wetzlar, Germany).