Fig 2: ECM stiffness regulates tip cell formation through PXN phosphorylation. (A) Western blots showed that ECM stiffening did not affect components of FAs but improved phosphorylation levels of FAK and PXN. (B) Immunocytochemistry showed that matrix stiffening increased p-PXN intensity, particularly at peripheral areas of hUVECs. (Scale bar: left 75 µm, right 25 µm) (C) Confirmation of adenovirus transfection. PXN overexpression dramatically enhanced the cellular stiffness (D), expression of tip cell enriched genes (E) and sprouting angiogenesis (F) of hUVECs, which were abolished by the mutant. (Scale bar: 200 µm) (G) Quantification analysis of relative invasion distance and sprout numbers in (F). (*p < 0.05).

Fig 3: Kindlin binds and recruits paxillin to NAs.(A) GFP-IP of lysates from HEK 293T cells overexpressing GFP-tagged paxillin, Hic5 and leupaxin constructs (Pxn, paxillin; Hic5; Lpx, leupaxin) and K2flag reveal interaction of kindlin-2 with all three paxillin family members. (B) GFP-IP of lysates from HEK 293T cells overexpressing GFP-tagged paxillin truncation mutants and K2flag identifies the paxillin LIM3 domain as kindlin-2-binding domain. (C) GFP-IP of lysates from HEK 293T cells overexpressing GFP-tagged kindlin-2 truncation/deletion mutants and Cherry-tagged paxillin (PxnCH) identifies the kindlin-2 PH domain as paxillin binding domain. (D) Co-IP of endogenous paxillin and kindlin-2 from KindCtr cells. (E) Purified His-tagged paxillin-LIM3 domain pulls down recombinant kindlin-2 in a Zn2+-dependent manner. (F) K2GFP and K2ΔPHGFP expressing KindKo cells seeded on FN for the indicated times and stained for paxillin and β1 integrin. (G) Fluorescence intensity line scans from K2GFP- (n=11 cells) and K2ΔPHGFP- (n=17 cells) expressing KindKo cells cultured on FN for 10 min and stained for paxillin and β1 integrin; error bars indicate standard error of the mean. Bar, 10 µm. EDTA, ethylenediaminetetraacetic acid; FN, fibronection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; ILK, integrin-linked kinase; IP, immunoprecipitation; K2GFP, green fluorescent protein-tagged kindlin-2; LIM, Lin-11, Isl-1 and Mec-3; NAs, nascent adhesions; PH, pleckstrin homology.DOI: http://dx.doi.org/10.7554/eLife.10130.018

Fig 4: Paxillin and its phosphorylation at Ser-272 are important for smooth muscle cell division. (A) Plk1-interacting proteins were precipitated with the GST-tagged Plk1 catalytic domain (GST-CD), and separated by SDS-PAGE. In separate experiment, precipitated proteins were analyzed by immunoblot analysis. Paxillin is one of the major Plk1-interacting proteins in HASM cells. The images are representative of three experiments. (B) Extracts of HASM cells were immunoprecipitated with paxillin antibody and blotted with paxillin antibody followed by membrane stripping. The membranes were reblotted with Plk1 antibody. Blots for input are also shown. Blots are representative of three experiments. (C) Representative immunoblots illustrating paxillin (Pax) knockdown (KD) and recombinant protein expression in cells. Blots of HASM cells transfected with control (Ctrl) siRNA or Pax siRNA with or without the DNA constructs for 2 days were probed with antibodies against Pax, phospho-paxillin (Ser-272) and GAPDH. Ratios of Pax/p-Pax over GAPDH in Pax KD, rescue, and S272A cells are normalized to ratios obtained from cells treated with Ctrl siRNA. WT, wild type Pax. Data are mean ± SE (n = 4). *P < 0.05. NS, not significant. (D) Cells were plated on 6-well dishes and cultured for 5 hours to allow for cell attachment. Cell division was monitored live using a time-lapse microscope for additional 24 hours. The time from round shape to cytokinetic abscission is increased in paxillin KD cells and cells expressing S272A paxillin, but restored in rescue cells. Data are mean ± SE, n = 32–39 cells. *P < 0.05. One-way analysis of variance was used for statistical test.

Fig 5: Loss of SNAP29 affects the pattern of focal adhesion complexes.(A–L) Control (SV80 and F572T) and CEDNIK (F110T or F165T) fibroblasts were fixed and stained with anti-FAK (A–C), anti-p-FAK (D-F), anti-PAX (G–I) and anti-p-PAX antibodies (J–L). Nuclei were stained with Hoechst. Bar, 10 µm. (M) Western blot analysis showing the expression of FAK, p-FAK, PAX and p-PAX in control (F572T) and CEDNIK (F165T, F110T) cells. Anti-ERK antibodies were used as a loading control. The blot is a representative of 5 independent experiments. (N) Bar graph illustrating the expression levels of the focal adhesion components: FAK, p-FAK, PAX and p-PAX. Densitometry was used to quantify the amount of each focal adhesion component as seen in Western blot, using the Image Densitometer 1Dprime. To normalize the results, the intensity of each band was divided by the intensity of ERK in the same lane. The value obtained for control cells (F572T) was considered as 100%. (O) Bar graph showing quantitation of the intensity of FAK, p-FAK, PAX and p-PAX staining, measured in 14-168 cells, using the ImageJ software. The value obtained for control cells (SV80) was considered as 100%. Expression pattern of FAK, phospho-FAK, paxillin and phospho-paxillin is different in CEDNIK cells in comparison to control cells. However, their total level is similar.