Mouse anti perk

A custom rabbit anti-heparanase 2 antibody was designed and manufactured (Generon). The immunizing peptide sequence used was QLDPSIIHDGWLDC (Fig. 1). Commercially available antibodies used in this study were: mouse anti-acetylated tubulin (Sigma), mouse anti-12/101 (Developmental Studies Hybridoma Bank), rabbit anti-ERK (Cell Signalling), mouse anti-pERK (Sigma–Aldrich), mouse anti-Na,K-ATPase (Abcam), rabbit anti-LRIG2 (Abgent), horseradish peroxidase (HRP)-coupled anti-mouse and anti-rabbit (Life Technologies). To test specificity, anti-heparanase 2 antibody was incubated overnight at 4°C on a rotator with a 50-fold excess immunizing peptide or a control reaction of 50-fold excess non-specific peptide, prior to use in IHC applications. Western blotting was performed by standard methods. Total protein was extracted from pools of three embryos, with amounts equivalent to one embryo separated via SDS–PAGE.

ARO cells were plated in 10-cm plates (8x10⁵ cells) and grown for 24 h. Cells were then treated either with 0.35% MCP, 75 μM FTS, 0.35% MCP plus 75 μM FTS or as a control with 0.35% D-lactose for 48 h. In a second set of experiments, cells were treated either with 0.35% MCP, 75 μM FTS, 0.35% MCP plus 75 μM FTS or as a control with 0.35% D-lactose for 72 h. Next, cells were lysed in 300 μl homogenization buffer (50 mmol/l Tris-HCl—pH 7.6, 20 mM MgCl₂, 200 mM NaCl, 0.5% NP40, 1 mM DTT, and protease inhibitors), centrifuged for 10 min at 14 000 r.p.m. at 4 °C and the supernatant was collected. Equal amounts of proteins (40 μg per lane) were subjected to SDS-PAGE, followed by immunoblotting with mouse anti-pan-Ras antibody (Ab, Calbiochem, San Diego, CA, USA), rabbit anti-β tubulin Ab (Sigma Aldrich, Rehovot, IL, USA), mouse anti- K-Ras (Calbiochem), rat anti-Galectin-3 (Mac2), rabbit anti-total ERK (Santa Cruz, Dallas, TX, USA), mouse anti-p-ERK (Sigma Aldrich), rabbit anti-p21 (Santa Cruz), mouse anti-p53 (Calbiochem) and mouse anti-actin (MP Biomedicals, Santa Ana, CA, USA). Blots were then exposed to the appropriate secondary peroxidase-coupled IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and subjected to enhanced chemiluminescence. Protein bands were quantified by densitometry with Image EZQuant-Gel software (EZQuant Ltd, Tel-Aviv, Israel).