Figure 2. POLH-1 is Destroyed in a PIP box and CRL4-Cdt2 Dependent Manner in MMS-exposed, GEI-17 Deficient Embryos.

(A) A PIP box deletion mutant (ΔPIP) was introduced into the pie-1::polh-1-GFP construct and stable transformants were obtained via particle bombardment (see Experimental Procedures). Early embryo extracts were prepared, and the WT POLH-1-GFP or ΔPIP POLH-1-GFP proteins were probed by immunoblotting, after SDS-PAGE, with anti-GFP antibodies. The lysates were also probed with antibodies against PCN-1, to ensure equal loading of the samples, and with anti-GEI-17 antibodies, to monitor the effectiveness of the gei-17 RNAi. The animals were exposed, or not, to MMS, as in Figure 1B.

(B) WT POLH-1-GFP worms were treated with gei-17/pcn-1 RNAi by soaking, as indicated, and then optionally exposed to MMS (as in Figure 1B). Early embryo extracts were prepared and the POLH-1-GFP protein was probed by immunoblotting, after SDS-PAGE, with anti-GFP antibodies. The lysates were also probed with antibodies against PCN-1 or GEI-17, to monitor the effectiveness of the gei-17/pcn-1 RNAi, as indicated. The intensity of the signals in the POLH-1-GFP blot were quantified using the histogram tool of Photoshop, and the vales obtained were 52, 51, 49.5, and 51, respectively, for the samples present in lanes 1–4 of the blot.

(C) Feeding RNAi, in various configurations, was performed on the WT POLH-1-GFP worm as indicated. The animals were optionally exposed to MMS (as in Figure 1B) and then embryo extracts were prepared. Extracts were fractionated on SDS-PAGE gels and then probed by immunoblotting for POLH-1-GFP (using anti-GFP antibodies) and GEI-17 and PCN-1, as indicated.

(D) Early embryo extracts were prepared and POLH-1, GEI-17, or PCN-1 proteins were then detected by immunoblotting, after SDS-PAGE. Prior to extract preparation, the animals were either treated, or not, with gei-17 RNAi by feeding and MMS (as in Figure 1B), as indicated.