Ab180760

The cells were lysed in radioimmunoprecipitation assay buffer (containing 1% protease and phosphatase inhibitor), and the total protein content was quantitatively measured using the bicinchoninic acid assay. Protein (35 μg) was separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred onto a polyvinylidene fluoride membrane, blocked with 5% non-fat milk for 2 h, and incubated with the primary antibody overnight at 4°C. After washing the polyvinylidene fluoride membrane using tris-buffered saline with 0.1% tween, immunoreactivity was measured using rabbit HRP-conjugated secondary antibody (1:5000; Millipore), and the beta-actin level was measured as a control. The following primary antibodies were used: anti-SOCS3 (1:1000, 52113 s), anti-p38 (1:1000, 8690 s), and anti-ASK1 (1:1000, 8662 s) from Cell Signaling Technology (Beverly, MA, USA); anti-TLR2 (1:1000, ab16894) anti-LL37 (1:1000, ab180760) from Abcam (Cambridge, MA); anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Imaging was performed using a ChemiDoc^TM XRS+ system (Bio-Rad, USA) and an HRP substrate (Luminata; Millipore), and the bands on the western blots were scored by scanning and quantitatively analyzing grayscale values using ImageJ (National Institutes of Health, Bethesda, MD).

Immunocytochemical staining of MSC for expression of antimicrobial peptides was performed as described previously (9 (link), 16 (link)). Briefly, 1 X 10⁴ cells were seeded on round coverslips (Chemglass Life Sciences, Vineland, NJ) placed within 24-well-cell culture plates overnight, then fixed with 4% paraformaldehyde (Fisher Scientific, Hampton, New Hampshire) for 10 min, washed with PBS and permeabilized with 0.1% Triton X-100(Sigma-Aldrich,). Slides were then blocked using 5% v/v normal donkey serum and then incubated with primary antibodies, diluted appropriately. Antibodies used for these studies included surfactant protein D antibody (ab203309), lipocalin-2 antibody (ab63929), beta 2 defensin antibody (ab9871), hepcidin antibody (ab134790), and cathelicidin antibody (ab180760), all obtained from Abcam (Cambridge, MA). Specificity controls for immunostaining included purified IgG antibodies from non-immune rabbits or goats. Following primary antibody incubation, slides were washed and incubated with secondary antibodies, either donkey anti-rabbit or donkey anti-goat, both conjugated to Cy3 (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pennsylvania) and the slides were then counter-stained with DAPI to visualize cell nuclei. Image capture for fluorescence staining was done using an Olympus IX83 spinning disk confocal microscope.