FIGURE 2.

Erk kinases mediates phosphorylation of Ser-200. A, HeLa cells were transfected with 1 μg of HA-h-Lin28a. 36 h after transfection, cells were pretreated with various kinase inhibitors before being harvested for HA immunoprecipitation (IP) and whole-cell lysates (Input). Then IP and input were detected by the indicated antibodies. MAPK-ERK inhibitors AZD6244 (10 μm) and U0126 (10 μm) and CDK4/6 inhibitor PD0332991 (1 μm) were used as indicated. B, HeLa cells were co-transfected with 1 μg of HA-h-Lin28a and 3 μg of shRNAs against ERK1/2. 36 h after transfection, cells were harvested for HA immunoprecipitation (IP) and whole-cell lysates (Input). Then IP and input were detected by the indicated antibodies. C, immunoblotting (IB) analysis of endogenous Lin28a Ser-200 phosphorylation in P19 cells after treatment with various kinase inhibitors. P19 cells were serum-starved for 18 h before the addition of kinase inhibitors and 10% serum. 5 h later, cells were harvested to prepare whole-cell lysates and detected by the indicated antibodies. MAPK-ERK inhibitors AZD6244 (10 μm) and U0126 (10 μm) and CDK4/6 inhibitor PD0332991 (1 μm) were used as indicated. D, P19 cells were co-transfected with 1 μg of mouse Lin28a constructs, wild type (HA-m-Lin28a) or phospho-deficient (HA-m-Lin28a-S200A), and with 2 μg of constitutively active HA-MEK1 (HA-MEK1 CA) construct. 36 h after transfection, cells were harvested for FLAG immunoprecipitation (IP) and whole-cell lysates (Input). Then IP and input were detected by the indicated antibodies. E, P19 and S200A (SA) knock-in cells were serum-starved for 18 h and then split into bacterial grade Petri dish with medium containing 10% serum, retinoic acid, and U0126 (10 μm) as indicated. 24 h later, cells were harvested and detected by the indicated antibodies. F, P19 and S200A knock-in cells were transfected using Lipofectamine with 2 μg of constitutively active HA-MEK1 construct. 24 h after transfection, cells were split into a bacterial grade Petri dish and induced with RA for another 24 h. Then cells were harvested and signal was detected by the indicated antibodies. G, ERK1 phosphorylates Lin28a Ser 200 in vitro. HA-ERK1 kinases were prepared by immunoprecipitation from 293T cells transfected with 2 μg of HA-ERK1 construct. 2 μg of wild type Lin28a or Lin28a-S200A recombinant proteins were incubated with HA-ERK1 kinase in the presence of ATP. 30 min later, the kinase reactions were stopped by the addition of the SDS sample buffer. The reaction products were resolved by SDS-PAGE, and phosphorylation of Lin28a was detected with CS2 motif antibody.