Anti myc antibody

COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum. Purified plasmids (0.1 μg pTarget-Myc-αA-crystallin, 0.1 μg pTarget-Myc-αB-crystallin and 1.5 μg pTarget-FLAG-FYCO1) were transfected into COS-7 cells using Lipofectamine LTX (Invitrogen) according to the manufacturer’s protocols. After 72 h cells were harvested and lysed with 20 mM Tris–HCl (pH7.4), containing 150 mM NaCl, 5 mM NaF, 1 mM Na₃VO₄, 500 µM EDTA, 200 µM AEBSF, 160 nM aprotinin, 10 µM bestatin, 3 µM E-64, 4 µM leupeptin, 2 µM pepstatin A, and 0.5% Triton X-100. After centrifugation at 19,000 × g, for 60 min at 4℃, the protein concentration was determined using a Bradford assay (Bio-Rad Laboratories, Inc.).
Immunoprecipitation was performed using 100 µg protein lysates. Lysates were incubated with protein G-agarose beads at 4℃ overnight with agitation and subjected to immunoprecipitation with 2 μg anti-Myc antibody (Santa Cruz Biotechnology, 9E10). Protein G–agarose beads were washed four times in cold RIPA buffer. Sample buffer (50 mM Tris–HCl, pH6.8, 2% SDS, 6% 2-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue) was added to Protein G–agarose beads and heated at 95 ℃ for 5 min. Samples were then analyzed by western blotting using the anti-Myc antibody (Santa Cruz Biotechnology, 9E10, 1:2,000) or anti-FLAG antibody (Sigma, M2, 1:8,000).

HEK293T cells were seeded at 3.10⁵ cells per well in 6-wells slides (Millicell, Millipore) precoated with poly-L-lysine (Sigma) and transfected with pcdna3-myc-MITF and pcdna3-RAF plasmids (HA-ARAF, HA-BRAF or HA-CRAF) or empty vector as previously described. After 48 hours, cells were fixed in 4% paraformaldehyde, blocked (0.1% Triton X-100, 10% goat serum in PBS) and stained overnight at 4 °C with anti-myc antibody (Santa Cruz) and anti-ARAF (Cell Signaling) or anti-MITF (Sigma) and anti-BRAF (Santa Cruz) or anti-CRAF (BD Biosciences). Anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 or anti-rabbit Alexa Fluor 594 and anti-mouse Alexa Fluor 488 were used for detection. Fluoroshield with DAPI (Sigma) was used as mounting medium. Images were captured using a 3D/optigrid Leica fluorescent microscope. For quantification by using Image J software, the nuclear and cytosolic compartments were selected by applying an automatic threshold (Li Dark method) to the DAPI and FITC images. The nucleus-cytoplasm ratio was then computed by dividing the mean intensity of TexasRed2 (MITF) fluorescence extracted from nucleus region by the mean intensity from cytosolic regions obtained by subtracting DAPI from the FITC surface. The background intensity was measured on each TexasRed2 image and subtracted from the mean intensities before calculating the ratio.

HEK293T cells were transfected with different plasmids using Lipofectamine 2000 (Invitrogen) and cultured for 24 hours. The cells were then washed with phosphate-buffered saline (PBS) at room temperature and lysed in ice-cold lysis buffer composed of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1 X protease inhibitor cocktail (Sigma-Aldrich). Monoclonal anti-myc antibody agarose beads were used for immunoprecipitation according to the manufacturer’s recommendation (Pierce). After 2 hours of incubation at 4 °C, immunoprecipitated proteins were washed five times with high salt lysis buffer (500 mM NaCl) and separated by polyacrylamide gel electrophoresis, then transferred to nitrocellulose membrane, probed with anti-GFP antibody (Roche) or anti-myc antibody (Santa Cruz Biotechnology) and detected with an Odyssey Infrared Imaging System (LI-COR Biosciences).