Anti α5β1 integrin

Cells cultured under different glucose conditions were washed once with PBS containing 0.04% EDTA and collected using cell dissociation solution (Sigma). Cells were washed once with DMEM containing 10% FBS and blocked in TBS with 1% goat serum for 20 min on ice. Cells were incubated with specific primary antibodies for 30 min on ice. The anti-cleaved caspase 3 antibody (Cell signaling, Danvers, MA), anti-GFAP (Dako, Carpentaria, CA), anti-α5β1 integrin (MAB1976Z), or anti-αvβ3 integrin (MAB1999) (Millipore, Temecula, CA) was prepared in TBS with 1% BSA at 2 µg/ml. Following incubation, cells were then washed twice with TBS with 1% BSA, and incubated with appropriate FITC-conjugated secondary antibody for 30 min on ice. The stained cells were washed twice with TBS with 1% BSA and resuspended in 0.5 ml of TBS with 1% BSA, and analyzed by FACScan caliber flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). Cells incubated with secondary antibodies in the absence of primary antibodies were used as negative control. The average mean fluorescence intensities were used for quantitative comparisons.

Primary antibodies for anti-CD44 (R&D systems, UK), anti-cortactin (Upstate Biotechnology, NY, USA), anti-paxillin, anti-phosphoTyr¹¹⁸-paxillin were obtained from Cell Signaling Technology (Beverly, MA), while anti-α5β1-integrin, anti-α2β1-integrin, anti-β1-integrin, activated-β1-integrin conformations (B44 and HUTS-4), cellular Fibronectin and phosphoTyr⁴²¹-cortactin were purchased from Millipore (Watford, UK). HRP-conjugated secondary antibodies were obtained from Amersham, UK and Hyaluronan (MW220 kDa) from Lifecore Biomedical (MN, USA). Cells were stimulated with 100 μg/ml HA for indicated times. All other reagents were purchased from Sigma (Poole, UK) unless otherwise stated.

LPS (from E. coli 0111:B4) and sodium octanoate (NaO) were acquired from Sigma-Aldrich (St. Louis, MO, USA). In this study, we used 0.25 and 1 mM NaO, which induces or inhibits S. aureus internalization into bMECs, respectively (Alva-Murillo et al., 2013 (link)). The monoclonal blocking antibodies used were anti-α5β1 integrin (Millipore Cat# MAB2514 Lot# RRID:AB_94626), anti-TLR2 (TL2.1, Abcam Cat# ab9100 Lot# RRID:AB_307008), and anti-CD36 (FA6-152, Abcam Cat# ab17044 Lot# RRID:AB_443600). The MAPK inhibitors SB20358 (p38), SP600125 (JNK), and U0126 (ERK1/2) were acquired from Cell Signaling Technology® (Boston, MA). The working solutions were dissolved in dimethyl sulfoxide (DMSO), which was employed as vehicle in the MAPK activation assay.