Quantitative pulldown with APD-CLD probe identifies covalent binding targets.
Figure 2b: PANC-1 cells were treated with an APD-CLD alkyne probe or an APD-CLD-deficient alkyne probe (125-1000 nM, 2-fold dilution) for 12 hours under hypoxia. The lysates were tagged with rhodamine-N3 via CuAAC, separated by electrophoresis and visualized by excitation of rhodamine B. Figure 2c: Protein targets in living PANC-1 cells were enriched by CuAAC to biotin-PEG3-N3 followed by pulldown on streptavidin-coated Dynabeads. The enriched proteins were separated by electrophoresis and visualized by SimplyBlue Safe Stain. Figure S2a: In-gel fluorescence analysis of covalent binding partners for the APD-CLD alkyne probe. PANC-1 cells were treated with siRNA against RTN3, RTN4, CYB5B or non-targeting control siRNA, and subsequently treated with an APD-CLD alkyne probe for 12 hours under hypoxia. Then cells were lysed and lysates were tagged by CuAAC to rhodamine B azide. The proteins were separated by SDS-PAGE and analyzed for fluorescence in a CCD camera. Subsequently, the gel was stained by SimplyBlue SafeStain for total protein visualisation. Figure S2c,d,e,f: See [Viability data.xlsx]. Cells were treated with either the APD-CLD alkyne probe or electrophilic acrylamide RTN4 ligands for 48 hours under normoxia or hypoxia. Viability was assessed by CellTIter Blue (Promega) and is given as RFUs.