Co-immunoprecipitation, western blot, and pull-down assay

Cells were lysed in NETN100 buffer containing protease inhibitor cocktails. Supernatants were collected by centrifugation for 5 min at 13,000 rpm and incubated with antibody and beads for 2 h at 4 °C. The immune-complex was washed followed by resolving in the SDS-PAGE for western blot analysis.

To test whether the ADP-ribosylation of Pol III is covalent or non-covalent, extraction of ADP-ribosylated proteins for immunoprecipitation analyses were modified as described in a previous study⁶⁸. Briefly, cells were lysed in SDS lysis buffer (1% SDS, 10 mM HEPES, pH 7.0, 2 mM MgCl₂, 500 U Benzonase, pH 8.5). Lysates were diluted with PBS to 0.2% SDS and mixed with antibody and beads for 2 h at 4 °C. Then the beads were washed with the SDS wash buffer (1% SDS, 100 mM HEPES, pH 8.5, 150 mM NaCl) and subsequently with the wash buffer (100 mM HEPES, pH 8.5, 150 mM NaCl). The ADP-ribosylated proteins were incubated with 2× SDS-PAGE sample loading buffer at 95 °C for 5 min and the eluates were subjected to immunoblotting analyses. To further confirm that ADP-ribosylation of Pol III is covalent, the 0.5 M NH₂OH treatment at room temperature on an end-to-end rotator was performed to detach PAR chain from Pol III proteins before signal detection.