Monoclonal anti gapdh

The U87-MG, U251-MG, and U138-MG cell lines were harvested and acid-treated, as described [41 (link)]. Then, 2 × 10⁶ cells/sample were exposed for 30 min at 4 °C to uPAcyclin or S-uPAcyclin at the indicated concentrations, or with the following antibodies: VNR147 monoclonal anti-αV integrin (Chemicon Int. Inc., Temecula, CA, USA), polyclonal anti-αV (Millipore, Burlington, MA, USA), monoclonal anti-α2 (Chemicon Int. Inc., Temecula, CA, USA), polyclonal anti-actin (Sigma-Aldrich, St. Louis, MO, USA), monoclonal anti-GAPDH (Abcam, Cambridge, UK), and purified vitronectin (VN, Corning, Glendale, AZ, USA). The cells were subsequently incubated with 50 nM FITC-uPAcyclin for 2 h at 4 °C, as described [25 (link)]. At the end of incubation, the cells were washed and the cell surface-associated fluorescence was determined using a fluorimeter (VICTOR^TM X3-PerkinElmer, Waltham, MA, USA). All binding assays were performed three times, with duplicate samples, and analyzed as described in Section 2.11.

The cells were lysed in RIPA Lysis Buffer (Beyotime, China) supplemented with protease inhibitors. The total protein concentration was measured using a BCA protein assay kit (Pierce, MA, USA) according to the manufacturer's instruction. A total of 20 μg protein was separated on 10% SDS-PAGE and transferred onto PVDF membranes (Beyotime). Blocking was performed in 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 2 hours. The membranes were incubated overnight at 4°C with primary antibodies, including the following: polyclonal anti-CXCR7 (1 : 400; Abcam, Cambridge, MA, USA), polyclonal anti-pan-AKT (1 : 1000; Abcam), polyclonal anti-phospho-AKT (1 : 500; Abcam), monoclonal anti-ERK1/2 (1 : 5000; Abcam), monoclonal anti-phospho-ERK1/2 (1 : 1000; Abcam), monoclonal anti-β-catenin (1 : 400; Abcam), monoclonal anti-c-Myc (1 : 500; Abcam), polyclonal anti-cyclinD1 (1 : 1000; Abcam), polyclonal anti-survivin (1 : 5000; Abcam), monoclonal anti-α-SMA (1 : 1000; Abcam), and monoclonal anti-GAPDH (1 : 10000; Abcam). The membranes were then washed with TBST three times, incubated with HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 2 hours at room temperature, then detected with ECL detection reagents (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as a loading control.