Fig 2: Expression levels of Lpd and associated actin regulators in Nestin-Cre-Lpd KO mice. Equal amounts of protein extracts (30 µg) of the cortices of adult Nestin-Cre-Lpd knockout (KO) and Lpd flox/flox control mice (WT) were separated by SDS-PAGE and analysed by Western blotting with antibodies against (a) Lpd, (b) Mena (note that three isoforms of Mena of 80, 88, and 140 kDa are expressed in adult brain33), (c) VASP (note that VASP is not expressed in adult brain33), (d) Scar/WAVE1, and (e) Scar/WAVE2. HSC70 re-probe of each blot serves as the loading control (note that the upper faint band in the HSC70 blot in (d) represents the remaining Scar/WAVE1 band. Molecular weight markers are indicated in kDa.

Fig 3: RGMa is required for AJ stability and recruitment of the WRC and Arp2/3.(a) RGMa-siRNA induced the loss of junctional integrity (Ecad+, red). (b) Coexpression of zNeo-Myc (green; Ecad, red; merge, yellow) with RGMa-siRNA rescued junctional integrity. (c) Line-scan analysis: mean Ecad peak fluorescence intensity (FI) in cells coexpressing RGMa-siRNA and zNeo-Myc or RGMa-siRNA alone (n=150 junctions, mean±s.e.m., ****P<0.0001, one-way analysis of variance (ANOVA), Dunn's post hoc test). (d) WAVE2 (green) was not associated with Ecad (red) at the AJ in RGMa-siRNA-expressing cells. (e) Line-scan analysis: mean WAVE2 peak FI in RGMa-siRNA cells. (f) Arp3 (green) was not associated with Ecad (red) at the AJ in RGMa-siRNA-expressing cells. (g) Line-scan analysis: mean Arp3 peak FI in RGMa-siRNA cells. (e,g) n=150 junctions, mean s.e.m., ****P<0.0001, Student's t-test. Scale bars, 15 µm (a,b,d,f).

Fig 4: NHSL1 co-localises with Abi and the Scar/WAVE complex.a Still images from live-cell imaging showing NHSL1-EGFP co-expressed with mScarlet-I-tagged Abi1 and b Nap1 in B16-F1 cells. Representative images shown from three independent experiments. Scale bar represents 20 µm. Inset is a magnified view of the white box. Scale bar in inset a applies to both insets a, b: 1.25 µm. See also related Supplementary Movie 6. c–e Endogenous NHSL1 co-localises with Abi1 (c NHSL1 pAb) Scar/WAVE1 (d NHSL1 pAb), and Scar/WAVE2 (e NHSL1 mAb) at the very edge of lamellipodia in B16-F1 mouse melanoma cells. Scale bar in c applies also to d, e: 20 µm. Inset represents a magnified view of the white box. Scale bar in the inset in c applies also to inset for d, e: 20 µm. Representative images shown from three independent experiments. See Supplementary Fig. 8 for line scans of co-localisations in c and e.

Fig 5: Neogenin recruits WAVE2 and Arp2/3 to the AJ.(a) WAVE2 (green) was not associated with Ecad (red) at the AJ in Neo-siRNA-expressing cells. Line-scan analysis: (b) mean WAVE2 fluorescence intensity (FI) across AJs and (c) mean WAVE2 peak FI at junctions. (d) Arp3 (green) was not associated with Ecad (red) at the AJ. Line-scan analysis: (e) mean Arp3 FI across AJs and (f) mean Arp3 peak FI at junctions. (g) Coexpression of full-length Ecad (Ecad-GFP) did not rescue the Neo-siRNA phenotype. (h) Line-scan analysis: mean Ecad peak FI in cells coexpressing Neo-siRNA and Ecad-GFP. (b,c,e,f,h) n=150 junctions, mean±s.e.m., ****P<0.0001, Student's t-test. Scale bars, 7 µm (a,d); 15 µm (g).