Anxa1

After the specific time of incubation, DU145 and DU145R80 cells were fixed in p-formaldehyde (4% v/v in PBS) for 5 minutes. The cells were permeabilized in Triton X-100 (0.5% v/v in PBS) for 5 minutes, and then incubated in goat or donkey serum (20% v/v PBS) for 30 minutes, and with primary antibodies against ANXA1 (rabbit polyclonal; 1:100; Invitrogen), vimentin (mouse monoclonal; 1:500; Santa Cruz Biotechnology) and FAK (mouse monoclonal; 1:100; BD Transduction Laboratories), overnight at 4°C. After two washing steps with PBS, cells were incubated with anti-rabbit and / or anti-mouse AlexaFluor (488 and/or 555; 1:1000; Molecular Probes) for 2 hours at RT and then with FITC-conjugated anti-F-actin (5 μg/ml; Phalloidin-FITC, Sigma) for 30 minutes at RT in the dark. Hoechst 33342 (Molecular Probes) was used to detect nuclei. The coverslips were mounted in Mowiol (Mowiol 4–88, Sigma-Aldrich). A Zeiss LSM 710 Laser Scanning Microscope (Carl Zeiss MicroImaging GmbH) was used for data acquisition. Images were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH).

Protein expression was examined by SDS-PAGE, as described previously [20 (link)]. Briefly, total intracellular proteins were extracted from the cells by freeze/thawing in lysis buffer containing protease inhibitors. Protein content was estimated according to Biorad protein assay (BIO-RAD). A total of 20 µg of proteins were visualized using the chemioluminescence detection system (Amersham biosciences; Little Chalfont, UK) after incubation with rabbit polyclonal primary antibodies against ANXA1 (1:10,000; Invitrogen; Carlsbad, CA, USA) and calreticulin (1:1000; Elabscience; Houston, TX, USA), with mouse monoclonal primary antibodies against TSG101 (1:1000; ThermoFisher Scientific; Waltham, MA, USA) and anti-β actin (mouse monoclonal; 1:1000; clone AC15; A5441, Sigma-Aldrich). The blots were exposed and analysed to Las4000 (GE Healthcare Life Sciences).

Protein expression was examined by Western blot, as previously described [12 (link)]. Proteins were visualized using the chemioluminescence detection system (Amersham biosciences, Buckinghamshire, UK) after incubation with primary antibodies against rabbit polyclonal ANXA1 (1:10,000; Invitrogen, Carlsbad, CA, USA), MMP2 (1:1000; GeneTex, Irvine, CA, USA), and mouse monoclonal vimentin (1:1000; Santa Cruz Biotechnologies, Dallas, TX, USA), α-tubulin (1:1000; Sigma-Aldrich, St. Louis, MO, USA), and β-actin (1:1000; Sigma-Aldrich, St. Louis, MO, USA). The blots were exposed to Las4000 (GE Healthcare Life Sciences, Buckinghamshire, UK) and the relative band intensities were determined using ImageQuant software (GE Healthcare Life Sciences, Buckinghamshire, UK). Results were considered to be significant if p < 0.01.