Figure 2. Identification of the cellular targets of Ceg3.
(A) Detection of modified proteins by the ADPR-specific antibody. Lysates of HEK293T cells expressing 4xFlag-Ceg3 or 4xFlag-Ceg3E/A were probed with antibodies specific for ADPR-modification (left), the Flag tag (middle), or both (right). Note that the band indicated by a red arrow in samples expressing Ceg3 detected by the ADPR antibody represents its potential targets. (B) Substrate probing by immunoprecipitation (IP). Lysates of HEK293T cells transfected to express 4xFlag-Ceg3 or 4xFlag-Ceg3E/A were subjected to IP with beads coated with the Flag antibody and the products resolved by SDS-PAGE were detected by silver staining (left) or probed with the ADPR-specific antibody (right). Note the presence of a band in samples expressing Ceg3 but not Ceg3E/A when detected with the ADPR antibody (red arrows). (C) Enrichment of ADP-ribosylated proteins by Af1521 from cells transfected to express Ceg3. Lysates of HEK293T cells expressing 4xFlag-Ceg3 or 4xFlag-Ceg3E/A were incubated with beads coated with recombinant Af1521 and the pulldown products resolved by SDS-PAGE were detected by immunoblotting with the ADP-ribose antibody (left) or by silver staining (right) (red arrows). (D) Interactions between ANT1 and Ceg3. Lysates of HEK293T cells co-transfected to express 4xFlag-ANT1 and GFP-Ceg3 (or GFP) were subjected to IP with beads coated with the Flag antibody and bound proteins resolved by SDS-PAGE were detected with Flag and GFP antibodies, respectively. The expression of 4xFlag-ANT1, GFP-Ceg3, and GFP were similarly probed in total cell lysates as input. (E) Colocalization of Ceg3 and ANT1. HeLa cells transfected to express RFP-Ceg3 and GFP-ANT1 were fixed and analyzed. Images were acquired using a Zeiss LSM 880 confocal microscope. Scale bar, 5 μm. The colocalization of Ceg3 with ANT1 was quantitated by Pearson correlation coefficient with ImageJ.