C raf antibody

Antibodies for flow cytometric analysis, PE-conjugated CD38 (clone HIT2) and APC-conjugated CD11b (clone ICRF44) conjugated with allophycocyanin (APC), were from BD Biosciences (San Jose, CA, USA). SLP-76, Lyn, Fgr, Vav1, p-tyr, HRP anti-mouse and anti-rabbit antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-c-Cbl (clone C-15, catalogue number sc-170, lot H0414) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NUMB Antibody catalogue number (703078) was from Thermo Fisher Scientific (Waltham, MA, USA). The c-Raf antibody were from BD Biosciences (San Jose, CA, USA). Protease and phosphatase inhibitors were purchased from Sigma (St. Louis, MO, USA). Protein G magnetic beads used for immunoprecipitation were from Millipore (Billerica, MA, USA).

Thyroid cells were plated in T75 flasks and allowed to grow until 70% confluence. Complete media was replaced with 5% charcoal dextran-treated FBS-containing media and flasks were incubated for 24 hours. Cells were treated for an additional 24 hours with +/− 10 μM VMR. Cells were collected by gentle scraping, spun down, and subjected to 1% NP-40 lysis buffer on ice for 10 minutes with periodic mixing. Samples were centrifuged for 10 minutes at 14,000 rpm and 4°C and supernatants were collected. GammaBind Plus Sepharose beads (GE Healthcare) were washed 3 times with PBS. CRAF antibody (BD Bioscience) was added to beads and the mixture was incubated for 2 hours at 4°C with constant rotation. At the same time, 100 μg of sample lysate was also incubated for 2 hours at 4°C with constant rotation to pre-clear lysates. The beads coated with antibody were washed three times with PBS before the pre-cleared lysate was added to them. This mixture was incubated overnight in eppendorf tubes at 4°C with constant rotation. The tubes were centrifuged for 10 minutes at 14,000 rpm and beads were washed three times in 1% NP-40 lysis buffer. Loading buffer was added to the samples. Samples were boiled for ten minutes and spun down. Supernatants were subjected to 12% SDS-polyacrylamide gel electrophoresis as in our prior studies and probed for CRAF with primary antibody (Cell Signaling).