Stx12 and CCDC22 compete for binding to the VPS16B/VPS33B complex. (A) Top, cartoon depicting CCDC22 coiled-coil (CC) domain. Bottom, Twin Strep pulldown. Purified Twin Strep-VPS16B/His-VPS33B complex bound to Strep-Tactin beads (VV beads) was tested for its ability to pulldown (PD) purified GST (A, lane 1), GST-Stx12 SNARE (B, lane 2), GST-Stx12 Cytosolic domain (C, lane 3), His-CCDC22 CC (lane 4) or a combination of His-CCDC22 CC together with GST (lane 5), His-CCDC22 CC together with GST-Stx12 SNARE (lane 6) and His-CCDC22 CC together with GST-Stx12 Cytosolic domain (lane 7). Bound proteins were analyzed by SDS-PAGE (Coomassie) and immunoblotting (IB) using antibodies against CCDC22 and GST. (B) VV beads were incubated with a constant amount of His-CCDC22 CC and increasing amounts of GST-Stx12 SNARE. Bound proteins were analyzed by SDS-PAGE (Coomassie). The last lane to the right represents VV beads that were incubated with buffer only (no His-CCDC22 CC or GST-Stx12 SNARE). (C) His-CCDC22 CC band intensity quantification of the experiment shown in B (n = 2).