RBP CELF2 regulates acute leukemia development via FAT10-mTORC1 [RIP-seq]

We demonstrated that CELF2 regulates FAT10, and promotes mTORC1 signaling pathway activation in MLL-AF9-AML mice. Overall design: K562 cells with either expression of 3xflag-HA-CELF2 or empty vector were seeded in 10 cm dishes and harvested. 5 µg of CELF2 antibody (or rabbit IgG (Sigma-Aldrich, 12-370, 3202364) was conjugated to protein A/G magnetic beads by incubation for 4 h at 4°C, and then wash three times with wash buffer and incubated with pre-cleared nuclear extraction in lysis buffer at 4°C overnight. After washing with wash buffer for three times, beads were resuspended in 50 µl PBS, followed by DNA digestion at 37°C for 15 min. Input and co-immunoprecipitated RNAs were recovered by TRIzol, extraction was analyzed RNA-seq. Input VC represented the RNA of input in k562 empty vector cells. Input OE represented the RNA of input in k562 3xflag-HA-CELF2 cells. CELF2 IP1 and CELF2 IP2 represented the co-immunoprecipitated RNAs with CELF2 in k562 3xflag-HA-CELF2 cells, in two replicates of the experiment.