Syndecan 2

Primary antibodies were obtained from Abcam (SNAIL, N-cadherin; Cambridge, MA, USA), BD Transduction (E-cadherin; Franklin Lakes, NJ, USA), Santa Cruz Biotechnology (syndecan-1, Santa Cruz, CA, USA) and Contreras et al (24 (link)) (syndecan-2). Anti-rabbit secondary fluorescein-conjugated antibody, anti-mouse and anti-rabbit secondary peroxidase-conjugated antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

To investigate the effect of p17 on phosphorylation status of STAT1 and STAT3 total lysates were prepared by solubilization of cells in E1A lysis buffer (250 mM NaCl, 50 mM Hepes pH 7.0, 0.1% NP40, 50 mM EDTA) containing phosphatase and protease inhibitors. To investigate the effect of p17 on total protein expression CXCR2, syndecan-2 and tubulin, proteins were prepared for electro phoresis by boiling in 1X SDS Sample buffer (50 mM Tris HCl pH 6.8, 2.5% beta mercaptoethanol, 2% SDS, 10% glycerol). Following transfer to nitrocellulose membranes (Bio-Rad) proteins were detected with the following primary antibodies: CXCR2 (Santa Cruz – sc-7304), syndecan-2 (Santa Cruz – sc-365624), a-tubulin (Sigma – T 6074), phospho STAT1(tyr701) (Cell Signaling - #9171), total STAT1 (Cell Signaling - #9172), phospho STAT3(tyr705) (Santa Cruz – sc-7993) and total STAT3 (Santa Cruz – sc-8019). Primary antibodies were detected with the horseradish peroxidase (HRP)-labeled secondary antibodies. Proteins were visualized by SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions.