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. 2000 Jan;11(1):117–129. doi: 10.1091/mbc.11.1.117

Figure 3.

Figure 3

Characterization of LPP/VASP binding. (A) Preparation of VASP-enriched extracts. Total Vero cell extracts (lanes 1) or proteins bound to profilin-Sepharose after incubation with Vero cell extracts (lanes 2) were analyzed by SDS-PAGE and stained with Coomassie blue (left panel) or transferred onto a nitrocellulose membrane and stained with anti-VASP antibodies (right panel). The positions of molecular mass markers (kilodaltons) are shown on the left. (B) The N-terminal portion of LPP interacts with VASP in vitro. The profilin-bound proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. (Lanes 1–3) Identical membranes were incubated with myc-tagged GST-LPP3–414 (lane 1), with myc-tagged GST-LPP3–414 in the presence of a 100-fold molar excess of GST (lane 2), or without protein (lane 3) and then probed with anti-myc antibodies. (Lane 4) An identical membrane was probed directly with anti-VASP antibodies to indicate the positions of VASP bands. (Lane 5) A membrane was incubated with GST-ActA235–584 and then probed with anti-ActA3 (Golsteyn et al., 1997a). The positions of molecular mass markers (kilodaltons) are shown on the left. (C) VASP coimmunoprecipitates with LPP. Vero cell extracts were incubated with nonimmune (lane 1) or purified MP2 (lane 2) antibody. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting with mouse anti-VASP antibody. Total cell extracts were used to indicate the positions of VASP bands (lane 3). The positions of molecular mass markers (kilodaltons) are shown on the left. (D) Scheme of GFP-LPP chimera. GFP-LPP3–414-M is the fusion of Green Fluorescent Protein (GFP), the N-terminal part of LPP (amino acids 3–414), the myc tag, and the membrane anchor of ActA protein (amino acids 613–639). This ActA sequence is able to target proteins to the outer membrane of mitochondria. (E) Production of GFP-LPP3–414-M chimera in transiently transfected HeLa cells. Total extracts of nontransfected (lane 1) or transiently transfected HeLa cells producing GFP-LPP3–414-M (lane 2) were analyzed by SDS-PAGE and Western blotting with LPP2 antiserum. The antibody recognizes a band at 90 kDa that is not present in nontransfected cells. The arrow indicates the position of endogenous LPP. The positions of molecular mass markers (kilodaltons) are shown on the left. (F) LPP recruits VASP to an ectopic mitochondrial localization. HeLa cells transiently transfected to produce GFP-LPP3–414-M were fixed, permeabilized, and stained with anti-VASP antibody. Note that in addition to structures identified as mitochondria in separate experiments, VASP also localizes to focal adhesions, as in a nontransfected cell (inset). Bar, 20 μm.