Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasion.

doi: 10.1016/j.matbio.2019.04.002

Figure Lengend Snippet: Fig. 1. Mapping citrullination sites on human fibronectin. (A) Schematic overview of citrullination sites mapped onto human FN comprised of repetitive units of type I-III domains (depicted in different shapes). Tandem mass spectrometry was used to map the positions of citrullinated residues in purified plasma FN, which were either untreated or subjected to in vitro enzymatic citrullination by PAD2, PAD4, or both. Three previously reported sites (R1035, R1036, and R2356) that were also detected here are shown (residue labeled in brown) ((a) van Beer et al. 2012 [27]; (b) K. Sipila et al. 2017 [26]). (B) Three-dimensional structure of the 9th and 10th fibronectin type III domain (PDB 4LXO) highlighting residues previously shown to be essential for integrin binding (Redick et al., 2000 [28]) (Left). (Red) RGD and PHSRN sequences essential for synergistic integrin binding. (Cyan) residues with the greatest degree of binding influence outside the RGD site (R1410 and R1476). (Yellow) residues that help to facilitate PHSRN interactions. (Right) MS-identified citrullination sites within the 9-10FNIII domains showing overlap with integrin binding residues R1410 and R1476, as well as additional sites near the integrin binding interface (R1434, R1479) and R1452 (underneath R1476 and not shown here).

Article Snippet: Knockdown of β1 integrin was carried out using β1 shRNA plasmids (sc-72028-SH) and β1 lentiviral particles (sc-72028V), or control shRNA lentiviral particles (sc-108080) from Santa Cruz.

Techniques: Mass Spectrometry, Purification, Clinical Proteomics, In Vitro, Residue, Labeling, Binding Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasion.

doi: 10.1016/j.matbio.2019.04.002

Figure Lengend Snippet: Fig. 4. β1-Dominant Integrin Expression on cFN promotes force-sensitive downstream signaling. The basic protocol for the force-inducible magnetic bead Co-IP experiments is shown in (A) with WB measurements for pFAK/FAK (n = 7), pSRC/SRC (n = 7), and pILK/ILK (n = 4), all normalized to the condition of FN-coated beads without force are shown in (B). Light brown background shading indicates beads exposed to magnetic force (+force) whereas white backgrounds indicate no magnetic force was applied (Representative blot in Suppl. Fig. 7C). Fluorescent signal from IF staining was quantified to reveal the total signal (C) and co-localization (D) within FAs of pFAK, pSrc, and vinculin (70 cells/substrate). Quantification of total pILK content per cell (E; n = 50 cells per substrate), total pGSK/GSK (F), and total nuclear pGSK/ cellular GSK (G) (n = 70 cells per substrate) as measured from confocal z-stacks of pILK- or pGSK/GSK-stained cells, as appropriate, shows enhancements of each metric on cFN compared to FN. All error bars represent min/max. Representative maximum intensity projections of pGSK-stained cells are shown in (H) with the p-GSK signal in yellow, nucleus outlined in red, and the cell edge in white. Results are non-significant unless otherwise indicated.

Article Snippet: Knockdown of β1 integrin was carried out using β1 shRNA plasmids (sc-72028-SH) and β1 lentiviral particles (sc-72028V), or control shRNA lentiviral particles (sc-108080) from Santa Cruz.

Techniques: Expressing, Co-Immunoprecipitation Assay, Staining