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. 2019 Jul 26;8:e49677. doi: 10.7554/eLife.49677

Figure 2. WAVE regulatory complex (WRC) and Arp2/3 interact with Non-stop.

Figure 2.

(A) Mass spectrometry analysis of Group 2 fractions revealed Arp2/3 and WRC complexes stably interact with Non-stop. None of these proteins were identified using vector only Control purifications. (B and C) Reciprocal pull-down verifies interaction between Non-stop and WRC subunit suppressor of extracellular cAMP receptor (cAR) (SCAR). (B) BG3 cells were transfected with an expression vector for Non-stop-2xFLAG-2xHA (Non-stop-FH) or no vector (Control). Recombinant Non-stop was immunoprecipitated using anti-FLAG affinity resin and purified interactors analyzed by immunoblotting for the presence of endogenous SCAR. Anti-HA immunoblots verified the presence of Non-stop-FH bait. NS marks a non-specific band. (C) Pull-down was performed as in B, but with SCAR-FLAG-HA (SCAR-FH) expression vector. Immunoprecipitated proteins were probed by immunoblotting to detect endogenous Non-stop. Anti-HA immunoblots verified the presence of SCAR-FH bait. (D) Endogenous Non-stop and SCAR occupy similar subcellular regions. Immunofluorescence of SCAR and Non-stop in BG3 cells. Cells were immunostained with anti-Non-stop (magenta), anti-SCAR (yellow), and DAPI (white). Scale bar is 10 µM. Inset is enlarged to the right. (E) Endogenous Non-stop and F-actin are found in similar subcellular regions. Representative images of BG3 cells immunostained with anti-Non-stop (magenta), phalloidin (F-actin)(yellow), and DAPI (DNA)(white). Scale bar is 10 µM. Inset is enlarged to the right. Images in D and E were adjusted with contrast limited adaptive histogram equalization using the ImageJ CLAHE algorithm (Zuiderveld, 1994).