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. Author manuscript; available in PMC: 2009 Dec 26.
Published in final edited form as: Mol Cell. 2008 Dec 26;32(6):757–766. doi: 10.1016/j.molcel.2008.11.016

Figure 1. GEI-17 Controls POLH-1 Protein Levels During the DNA Damage Response.

Figure 1

(A) Early embryos were obtained by bleaching gravid N2 adults and extracts were prepared as described (Kim et al., 2007). The samples were fractionated on SDS-PAGE and then probed, by immunoblotting, with antibodies against POLH-1. The lysates were also probed with antibodies against PCN-1, to ensure equal loading of the samples. Prior to extract preparation the animals were either treated, or not, with polh-1 RNAi by feeding, as indicated.

(B) Early embryo extracts were prepared and then probed, by immunoblotting, with anti-POLH-1 antibodies, after SDS-PAGE. The lysates were also probed with anti-PCN-1 antibodies, to ensure equal loading of the samples, and with anti-GEI-17 antibodies, to monitor the effectiveness of the gei-17 RNAi. Prior to extract preparation, the animals were either treated, or not, with gei-17 RNAi and MMS, as indicated. MMS treatment was performed by inclusion of 0.005% MMS in the plate media and incubation of animals on these plates for 20 hours. We note that POLH-1 runs as doublet on this blot; this was not reproducibly observed, and we suspect that the lower band in the doublet represents a proteolytic fragment of POLH-1 that is occasionally produced during preparation of the extracts.

(C) Early embryo extracts were prepared after optional gei-17 RNAi, and then probed, by immunoblotting, with antibodies against POLH-1, GEI-17, or PCN-1, after SDS-PAGE. Prior to extract preparation the animals were either exposed, or not, to 100 J/m2 of UV light, as indicated. The samples were harvested 5 hours after UV irradiation.

(D) A POLH-1-GFP fusion protein was expressed in animals via stable transformation with a construct driving expression of POLH-1-GFP with the pie-1 promoter (“pie-1::polh-1-GFP”, see Experimental Procedures). These animals were treated with the indicated RNAi and early embryos were examined for GFP signals by fluorescence microscopy. The samples were also stained with Hoechst 33258, to visualize the DNA. “GFP” indicates GFP-based fluorescence, “DNA” indicates Hoechst 33258-based fluorescence, and “merge” shows both signals together in the same image. The exposure times during image capture was identical for all of the samples shown. Where indicated, animals were treated with MMS, as in Figure 1B.

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