Erk1 2

At 48 hpi, cells were lysed, and 20 µg of protein was subjected to reducing discontinuous sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (12% lower gel and 4% stacking gel). Proteins were electrotransferred onto nitrocellulose membranes and probed for total Vav (rabbit anti-Vav, 59 ng/mL, 1/2000 dilution, Abcam, ab40875), Vav2 (rabbit anti-Vav2, 1 µg/mL, 1/1000 dilution, Invitrogen, SC68-03), and actin (mouse anti- actin, Clone 4, 200 ng/mL, 1/5,000 dilution, Millipore, MAB1501R). Bound antibody was detected using goat anti-rabbit and goat anti-mouse horseradish peroxidase conjugate at 1/25,000 dilution. Chemiluminescence was detected using Clarity Western ECL substrate (BioRad) with the acquisition of images on the ChemiDoc Touch imaging system (BioRad). For analysis of cell signaling pathways, membranes were probed in combination for phospho p44/42 MAPK (ERK1/2; New England Biolabs #9101S) and mouse anti-β-tubulin (Invitrogen #32–2600) or total p44/42 MAPK (ERK1/2; New England Biolabs #9102) and mouse anti-β-tubulin with co-detection with IRDye 800CW donkey anti rabbit IgG (Millenium Science 926–32213) and IRDye 680RD donkey anti mouse (Millenium Science 926-68072). Blots were scanned using an Odyssey CLx Imaging System (LI-COR Biosciences).

Proteins (20 µg) were separated by SDS-polyacrylamide gel electrophoresis and blotted to PVDF membranes (Bio-Rad, Munich, Germany). Membranes were incubated with antibodies in 1.5% BSA/PBS/0.1% Tween 20. Signals were detected with the ECL Western blot detection system (Amersham Pharmacia, Deisenhofen, Germany). E-cadherin, ERK1/2, GAPDH and p38 MAPK antibodies were from New England Biolabs GmbH (Frankfurt, Germany). Antibodies to detect human and murine chemerin were from R&D Systems (Wiesbaden-Nordenstadt, Germany). ImageJ software was used for quantification [48 (link)].