Huts 21

Two millions HeLa cells were seeded in a 10 cm diameter dish, left untreated, irradiated (8 Gy), or intoxicated with CDT (1μg/ml) for the indicated periods of time. Cells were then detached with 2.5 mM EDTA in PBS. Four hundred thousand cells were incubated with the primary antibody (diluted 1:50 in PBS) on ice for 45 minutes. As positive control for integrin activation, cells were incubated with the primary antibody in PBS without Ca²⁺ and Mg²⁺ containing 2 mM MnCl₂ as previously described [31 (link)]. As negative control, cells were incubated with isotype-matched immunoglobulins. After washing in PBS, cells were incubated with the Alexa Fluor 555 donkey anti-mouse secondary antibody (Life technologies) for 45 minutes on ice. Flow cytometry analysis was performed using a FACSCalibur (BD Bioscience, San Jose, CA, USA). Data from 1x10⁴ cells were collected and analyzed using the CellQuest Pro software (BD Bioscience).
The following monoclonal antibodies were used: the pan anti-integrin β1 4B7R (R&D Systems) and HUTS-21 (BD Bioscience) that recognizes the activated form of integrin β1 [31 (link)].

Antibodies against total integrin β1 and activated integrin β1 (HUTS-21) were purchased from BD Biosciences (Lincoln Park, NJ). Integrin β1 blocking antibody (P4C10) was purchased from Millipore (Billerica, MA). Antibodies against C1GALT1, GAPDH, and focal adhesion kinase (FAK) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against phospho (p)-FAK was purchased from Cell Signaling Technology Inc. (Beverly, MA). Antibody against actin was purchased from GeneTex Inc. (Irvine, CA). Vicia villosa agglutinin (VVA) and peanut agglutinin (PNA) lectins were purchased from Vector Laboratories (Burlingame, CA). Human collagen IV, human fibronectin, murine laminin, bovine serum albumin (BSA), and protein de-glycosylation kit were purchased from Sigma (St Louis, MO).