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. 2016 Mar 14;212(6):707–719. doi: 10.1083/jcb.201507054

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© 2016 Le et al.

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Figure 4. — ROBO1 colocalizes and complexes with CMA components. (A) Representative images of MECs from wild-type (+/+) and Robo1^−/− mice stained for FAK (red), ROBO1 (green), and Hoechst (blue). Magnified boxed are the regions of +/+ cells; arrowheads point to colocalization. Bars, 10 µm. (B) Quantification of FAK-labeled FAs in MECs (n = 10+ cells). (C) Representative images of NMuMG cells infected with Robo1 or scrambled (SCR) shRNA lentivirus and stained for GFP (green), phalloidin (red), and Hoechst (blue), and in next panel, pFAK397 (white). Inset: Magnified versions of boxed regions with dashed lines encircling FAs. (D) Quantification of the size of pFAK397-labeled puncta (n = 3 experiments). (E and F) Representative blots from column coimmunoprecipitation on HME50 (E) and NMuMG (F) using anti-PXN bait. Samples of flowthrough (FT), first and second wash (W1/W2), and first and second elutions (E1/E2) were blotted for PXN, FAK, ROBO1, and GAPDH. (G) Representative images of +/+ MECs stained for PXN (green), ROBO1 (red), and Hoechst (blue). Magnified boxed; arrowheads point to colocalization. Bars, 30 µm, unless labeled otherwise. Asterisks denote significance by t test: *, P < 0.05; **, P < 0.01.