FIGURE 4.

PPM1G regulates the amount of 4E-BP1 associated with the cap structure. A, knockdown of PPM1G decreases the amount of 4E-BP1 bound with the m⁷GTP cap complex. The stable control and PPM1G knockdown 293E cells were subjected to one of the following treatments: kept in serum-containing medium (+S), serum-starved overnight (−S), serum-starved overnight and subsequently treated with insulin (+Ins), and pretreated with rapamycin before adding insulin (+Rapa/Ins). Cell lysates were incubated with m⁷GTP-beads to pull down cap-associated proteins. The amount of proteins and phosphoproteins in precipitates and lysates were detected by immunoblotting analysis. B, the amount of 4E-BP1 associated with the m⁷GTP-beads was quantified and normalized to the amount of cap-bound eIF4E. This number was set to 1 for cells grown in serum-containing medium. Data shown in the graph represent the mean ± S.E. (n = 4). *, p < 0.01; #, p < 0.05 as determined by two-sample Student's t tests comparing PPM1G knockdown (Sh-PPM1G) HCT116 cells with stable control (sh-Con) cells under each condition.