Technical Reference #1746

1746.

Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages Athanassios Dovas; Jean-Claude Gevrey; Alberto Grossi; Haein Park; Wassim Abou-Kheir; Dianne Cox, Albert Einstein College of Medicine, Journal of Cell Science, 122(1746), (2009)
Link To Paper

Keywords:
WASp; Podosome; Phosphorylation

Materials & Methods:
Cells antibodies and reagents RAW/LR5 cells were derived from the murine monocyte-macrophage RAW 264.7 cell line and were grown in RPMI medium (Mediatech) containing 10% new born calf serum (Cambrex Walkersville MD) 100 U/ml penicillin and 100 g/ml streptomycin (Sigma). Human monocyte-derived macrophages were a gift from Indra Sethy-Coraci (Columbia University NY). All cells were maintained at 37°C in a 5% CO2 incubator. Recombinant mouse CSF-1 was purchased from R&D Systems (Minneapolis MN). Purified human plasma fibronectin was from Sigma whereas vitronectin was from Invitrogen (Carlsbad CA). Rabbit polyclonal antibodies against WASp (H-250) and Cdc42 (P1) were from Santa Cruz Biotechnology (Santa Cruz CA). Mouse monoclonal vinculin (hVIN-1) and b-actin (AC-15) antibodies were from Sigma. Mouse monoclonal Myc-tag antibody (9B11) was from Cell Signaling Technology (Beverly MA). Alexa Fluor 568-phalloidin and secondary antibodies conjugated to Alexa Fluor 488 or 568 used for immunostaining were from Molecular Probes (Eugene OR). All secondary antibodies conjugated to horseradish peroxidase (HRP used for western blotting) were from Jackson ImmunoResearch Laboratories (West Grove PA).

Microscopic Technique
Immunofluorescence Microscopy

Cell Type(s)
RAW/LR5 cells