Extended Data Fig. 7: Mapping the architecture of the shieldin complex.

a, Streptavidin pulldown analysis determining which region of SHLD2 associates with the other shieldin subunits. WCEs of 293T cells transfected with an expression vectors for Flag–SHLD1, V5–SHLD3, GFP–REV7 and Strep/HA-tagged SHLD2, SHLD2-N (residues 2–420), SHLD2-C (residues 421–904) or empty Strep/HA vector (EV) were incubated with streptavidin resin and bound proteins were eluted with biotin. WCEs and elutions were analysed by SDS–PAGE and immunoblotting with the indicated antibodies. Tubulin was used as a loading control. Results are representative set of immunoblots from two independent experiments. Asterisk denotes a non-specific band. b, Mapping the SHLD3 and REV7 binding sites on the SHLD2 N terminus through streptavidin pulldowns with different SHLD2 constructs (detailed in Extended Data Fig. 6a) and immunoblotting. Results are a representative of a set of immunoblots from three independent experiments. c, Affinity purification of shieldin complex components using N-terminally truncated SHLD2 (Δ1–50) analysed by immunoblotting (representative of three independent experiments). d, Streptavidin pulldown analysis of SHLD2 association with REV7 and SHLD3. 293T cells were transfected with siRNAs and expression vectors for epitope-tagged shieldin components as indicated (EV, empty Strep/HA vector). WCEs were incubated with streptavidin resin and bound proteins were eluted with biotin. WCEs and elutions were analysed by SDS–PAGE and immunoblotted with the indicated antibodies. Short and long exposures are shown for GFP and V5 immunoblots (n = 1). e, Dependency of V5–SHLD3 co-immunoprecipitation with GFP–REV7. 293T cells were transfected with siRNAs and expression vectors for epitope-tagged REV7 and SHLD3 as indicated (EV, empty V5 vector). WCEs were incubated with anti-V5 antibody and protein G resin. Bound proteins were boiled in SDS sample buffer and analysed by immunoblotting with GFP and V5 antibodies (n = 1). f, Association between SHLD3 and RIF1. WCEs of 293T cells transfected with an expression vector for unfused GFP (−) or GFP–SHLD3 (SHLD3) were incubated with GFP-Trap resin. Bound proteins were boiled in SDS sample buffer and analysed by SDS–PAGE and immunoblotting against 53BP1 and RIF1. Results are representative of 2 SHLD3 immunoprecipitations, using SHLD3 fused to GFP (shown here) and V5 (shown in Fig. 3g) affinity tags.