P paxillin

To visualize the actin cytoskeleton, we performed immunofluorescence staining. We seeded 2.5 × 10⁴ cells on glass cover slips and incubated overnight. We fixed cells with 4% formaldehyde for 15 minutes at room temperature (RT) and then washed three times with PBS for 5 minutes each. We permeabilized cells with ice-cold 100% methanol for 10 minutes at −20°C; washed with PBS for 5 minutes; and blocked with 10% goat serum for 1 hour at RT. Next, we incubated cells for 1 hour at RT with an antibody against phosphorylated paxillin (p-paxillin, Cell Signaling) in 5% goat serum. After incubation, we washed cells with PBS 3 times for 5 minutes each and co-stained with Alexa Fluor 488 conjugated secondary antibody (Jackson ImmunoResearch Laboratory) for p-paxillin and conjugated Texas Red-X phalloidin (ThermoFisher Scientific) for actin for 1 hour at RT in the dark. We washed the slides and mounted with medium containing an anti-fade reagent and DAPI (ProLong™ Gold, ThermoFisher Scientific). We acquired images on an Olympus IX73 microscope with a DP80 CCD camera (Olympus), and we analyzed epifluorescence images with cellSens software (Olympus) and stress fibers with in-house MATLAB code.

NL monocytes were untreated or treated with flagellin (100 ng/ml) for 15–65 min. Cell lysates were examined by Western blot analysis (14 (link), 15 (link)). Blots were probed with phospho (p)-AKT1, pERK, p-p38, pJNK, p-paxillin and pFAK (Cell Signaling; 1:1000 dilution) and IκB Abs (Santa Cruz; 1:3000 dilution) or probed with AKT, ERK, p38 or actin Abs (Cell Signaling or Sigma; 1:3000 dilution).

Total protein (40 μg) was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then blocked with 5% non-fat milk for 1 h, incubated with primary antibody overnight at 4 °C, washed thrice with PBS-T (PBS plus 0.1% Tween 20), and incubated with HRP-linked secondary antibody for 1 h at room temperature. The membrane was then washed and bands were visualized by chemiluminescence assay. The following antibodies were used: uPAR, cathepsin B, ERK, p-ERK, JNK, p-JNK, p38, p-p38, Vinculin, α-Actinin, Talin, PI3K, p-PI3K, Rac-1, MEKK-1, Laminin and GAPDH (all from SCBT, Santa Cruz, CA). We also used antibodies for Paxillin, p-Paxillin, Pak-1 and p-Pak-1 (all from Cell Signaling Technology, Danvers, MA). We obtained Ras10 from Millipore (Billerica, MA).
For immunoprecipitation, cell lysates (300 μg) were pre-cleared by protein A/G micro-beads (Miltenyi Biotec, Auburn, CA) and then incubated with specific antibodies at a dilution of 1:100 overnight at 4 °C. The beads were washed with lysis buffer and resuspended in sample buffer before the immunoprecipitated protein was subjected to immunoblotting.