Rabbit phospho mek1 2 ser217 221

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).

For determination of MAPK signaling, iMLL-ENL LICs were isolated and transduced with WT, C295 mutant MSN, or L61 HRAS. Two days after transduction, transduced cells were sorted and grown in vitro for 5 more days. A total of 10⁷ cells were lysed, cleared, and further processed as above. The following antibodies were used: rabbit β-actin (1:1000; Cell Signaling Technology, 4970; RRID: AB_2223172), rabbit p44/42 MAPK (Erk1/2) (1:1000; Cell Signaling Technology, 9102; RRID: AB_330744), rabbit phospho-p44/42 MAPK (Erk1/2) (Thr²⁰²/Tyr²⁰⁴) (1:1000; Cell Signaling Technology, 9101), rabbit MEK1/2 (1:1000; Cell Signaling Technology, 9122; RRID: AB_823567), and rabbit phospho-MEK1/2(Ser^217/221) (1:1000; Cell Signaling Technology, 9121). For determination of MSN protein level upon Msn shRNA knockdown, iMLL-ENL LICs were isolated and transduced with scramble or Msn shRNA. Two days after transduction, transduced cells were collected, lysed, and processed as above. Rabbit moesin (1:1000; Abcam, ab52490; RRID: AB_881245) was used.