Figure 5. CDTa-specific nanobodies inhibit the enzymatic activity of CDTa.

HEK cells were lysed with Triton-X-100 and cell lysates were clarified by centrifugation. Before addition to the lysates, CDTa was preincubated for 20 min with serial dilutions of CDTa-specific or control (co) nanobodies at the indicated molar ratios of VHH:CDTa. ³²P-NAD⁺ was added as a substrate for CDTa-catalyzed ADP-ribosylation of actin. Reactions were stopped after 15 min by addition of SDS-PAGE sample buffer. Proteins were size fractionated by SDS-PAGE and covalently incorporated radioactivity was detected by exposing the dried gel to an X-ray film.