Mouse anti braf

HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transient transfections were performed with Transfectin reagent (Bio-Rad, 1703352). The lung cancer cell lines A549 (ATCC CCL-185) and H1666 (ATCC CRL-5885) were grown in Roswell Park Memorial Institute media (RPMI), the melanoma cell line A2058 (ATCC CRC-11147) in DMEM. The media was supplemented with 10% FBS and 10 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (Hepes). Quail embryo fibroblasts (QEF) were grown in Avian cell culture medium. DNA transfection was mediated using the calcium phosphate method. Primary antibodies used were the mouse anti-Rluc antibody directed against Rluc-F[1] (Chemicon, #MAB4410), mouse anti–HA-tag (Covance, MMS-10P), mouse anti-BRAF (Santa Cruz, F-7: sc-5284), mouse anti-MEK1/2 (Cell Signaling, 4684S), rabbit phospho-MEK1/2 (Ser217/221) (Cell Signaling, 9154), and rabbit anti–P-ERK1/2 (Cell Signaling, 9101).

HEK293, A375, and SW480 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transient transfections were performed with TransFectin reagent (Bio-Rad, #1703352) or jetPRIME (Polyplus). Primary antibodies used were mouse anti-Rluc antibodies (Chemi-Con, #MAB4400 versus Rluc-F[2] and #MAB4410 versus Rluc-F[1]), mouse anti–HA-tag (Covance, #MMS-10P), mouse anti-FLAG (Sigma-Aldrich, #F3165), mouse anti-V5 (Invitrogen, #R9302), mouse anti-BRAF [Santa Cruz Biotechnology, sc-5284 (#F-7) and sc-166 (#C-19)], rabbit anti-CRAF (Cell Signaling Technology, #9422), mouse anti-RAS (Thermo Fisher Scientific, #MA1-012X), mouse anti-GFP (green fluorescent protein) (Roche, #11814460001), mouse anti-MEK1/2 (Cell Signaling Technology, #4684S), rabbit anti–P-MEK1/2 (Ser²¹⁷/Ser²²¹) (Cell Signaling Technology, #9154), rabbit anti–P-ERK1/2 (Cell Signaling Technology, #9101), rabbit anti-ERK1/2 (Cell Signaling Technology, #4696S), and mouse anti-PKA RIIβ (BD Biosciences, #61062).