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. 2013 Sep 27;32(20):2722–2734. doi: 10.1038/emboj.2013.212

Figure 4.

Figure 4

Lamellipodin is recruited to CCPs and interacts with the EGFR. (A) HeLa cells expressing mCherry-Lpd and EGFR-GFP were imaged using TIRFM. Single colour (magnified square) and merged images of one representative cell are shown. Scale bar: 30 μm (left image) and 5 μm (right image). (B) IP of EGFR from HEK-293 cells overexpressing EGFR-GFP using Lpd-specific antibodies or IgG control. EGFR was detected using anti-GFP antibodies (left panels). Reprobe of the same blot with Lpd-specific antibodies (right panels). (C, D) Co-IP of endogenous EGFR and Lpd from A431 cell lysate using Lpd (C) or EGFR-specific antibodies (D) or IgG control. EGFR and Lpd were detected using specific antibodies. (E, F) Co-IP of endogenous EGFR and Lpd from HeLa cell lysate using Lpd (E) and EGFR-specific antibodies (F) or IgG control. Cells were stimulated with 2 ng/ml EGF (+) for 5 min or not stimulated (−). (B–F) A representative blot each from at least three independent experiments is shown. (G, H) HeLa cells expressing mCherry-Lpd and (G) GFP-Lpd or (H) GFP-Mena and mRFP-Clc were imaged using TIRFM. Single colour (magnified square) and merged images of one representative cell are shown. (G, H) Scale bar: 30 μm (G) and 10 μm (H) (left image) and 5 μm (G) and 2 μm (H) (right image). (G, H) Quantification of the percentage of colocalization of mRFP-Clc with Lpd-GFP (Clathrin versus Lpd) (G) or GFP-Mena (Clathrin versus Mena) (H) and Lpd-GFP with mRFP-Clc (Lpd versus Clathrin) (G) or GFP-Mena (Mena versus Clathrin) (H). Each time point of TIRF movies from four cells were analysed containing on average 850 Clc-positive and 450 Lpd-positive spots each. (I, J) Dynamics of Lpd-GFP and mRFP-Clc in HeLa cells was assessed every 5 s using TIRFM. Single colour and merged images of an area of a representative cell are shown. Arrows show recruitment of Lpd-GFP to mRFP-Clc shortly before scission. Scale bar: 1 μm (see also Supplementary Movie S3). (J) Quantification of the percentage of scission events of CCPs containing mRFP-Clc and Lpd-GFP. In total, 700 scission events of 3 different cells were analysed for each experiment.