FIGURE 9.

Co-localization and physical interaction of PARP12 with members of the NF-κB signaling pathway. A, PARP12 possesses a putative RHIM motif. The sequence of PARP12 was aligned to the RHIM motifs of known RHIM-containing proteins using the ClustalW algorithm, and compared with the Pfam RHIM consensus motif. B, HeLa cells were transfected with YFP-tagged PARP12ΔZnF (green) and Flag-tagged TRIF, grown at 37 °C for 24 h, fixed, and permeabilized, and TRIF (red) was detected with a monoclonal anti-Flag antibody and secondary goat anti-mouse Alexa594. The merged images (right panel) show colocalization between PARP12ΔZnF and TRIF. B, HeLa cells were transfected with YFP-PARP12ΔZnF (green) and Flag-tagged RIP1, and RIP1 (red) was detected with M2 anti-Flag and anti-mouse Alexa594 antibodies. The images were acquired with a Zeiss LSM710 confocal microscope (63×). C, HEK293T cells were transfected with different constructs coding for myc-tagged PARP12 (WT, catalytic mutant, GW, zinc finger mutant, dZnF) and Flag-tagged TRIF. The cells were lysed and myc-PARP12 was immunoprecipitated using anti-myc microbeads (Miltenyi). TRIF was detected with the M2 anti-Flag antibody, and PARP12 was detected with the MP12.1 antibody. The spliced lanes were merged from a single immunoblot.