Mab330

mAbs reactive to ectodomain of human GPCRs were purchased from R&D system (MAB197, MAB330, MAB331, MAB190, MAB6594, MAB4139, MAB099, MAB4224, MAB8396, MAB42273, MAB150, MAB179, MAB8276, MAB2016, MAB8561, MAB145, MAB1567, MAB699, and MAB9305) except for P2RY13 which was from ProMab (30487). Anti-HSV-1 gD antibody DL6 was purchased from Santa Cruz Biotechnology (sc-21719) and ascities fluid for DL6 was a kind gift from David Johnson (OHSU). Anti-V5 antibody was purchased from Thermo Fisher Scientific (R960-25). Cy3-conjugated anti-mouse IgG was purchased from Jackson ImmunoResearch.

Anti-CXCR1 (MAB330, R&D Systems), anti-CXCR2 (MAB331-100, R&D Systems), and anti-IL-8 (AB-208-NA, R&D Systems) blocking antibodies were diluted to 50 µg/ml in PBS and added to osteoclast cultures daily.

MDA-MB 231 cells were plated into 24-well plates (10⁵ cells/well) in complete medium. Confluent monolayers were nutrient starved by growing them for 24 h in medium containing 0.5% FBS and then scratched using a 200 μl pipette tip. After washing, cells were treated or not with 10 ng/ml of refp17 or vp17s in complete medium (10% FBS). When reported, starved MDA-MB 231 cells were pretreated with 2.5 μg/ml of mAb to CXCR1 (mAb 330; R&D, Minneapolis, MN, USA) or to CXCR2 (mAb 331; R&D), or with an isotype-matched mAb (2.5 μg/ml; R&D) for 1 h at 37 °C before proteins stimulation. In some experiments, MDA-MB 231 cells were serum starved for 24 h in the presence or absence of inhibitors of PI3K/Akt (LY294002) (20 μM) (ENZO Life Sciences, Farmingdale, NY, USA), Jak/STAT (AG-490) (20 μM) (Sigma-Aldrich, St. Louis, MO, USA) or MEK/ERK1/2 (PD98059) (10 μM) (Calbiochem, Billerica, MA, USA) signaling pathways. Cell migration was evaluated at different time points using an inverted microscope (DM-IRB microscope system, Leica, Buffalo Grove, IL, USA). Cells were photographed using a CCD camera (Hitachi Inc., Krefeld, Germany). Wound closure was monitored over 12 h. In some experiments, in order to count the cells migrating into the wound area or protruding from the border of the wound, cells were fixed before wound closure and stained with Comassie brilliant blue.