Enhanced luminescence reagents

Proteins were separated by SDS/PAGE (10% or 13% gels) in a minigel apparatus (BioRad, Munich, Germany) and transferred to nitrocellulose membrane (HP40, Roth, Karlsruhe, Germany) [19 (link)]. Western blotting for the quantification of total or phosphorylated proteins was performed using (phospho-) specific antibodies, HRP-linked secondary antibodies (1:6000), and enhanced luminescence reagents (Biozym, Oldendorf, Germany). Signals were recorded using a Fusion FX7-SL gel imager (Vilbert Lourmat, Eberhardzell, Germany). Band signal intensities were assessed by densitometry using Phoretix 1 D (Nonlinear Dynamics, Newcastle upon Tyne, UK). To correct for potential minor differences in exposure time, the mean density of all bands on each gel image was used to normalize the densities of individual bands of the same gel. Signal intensities of the phosphorylated forms of proteins were normalized to the signals obtained using antibodies against the respective core proteins or against the β-actin band densities. Relative band densities were used to calculate means and standard deviations of different experiments.

Cylindrospermopsin was obtained from Enzo Life Sciences (Lörrach, Germany) and dissolved in 20% methanol in an aqueous solution (both HPLC grade). The stock (1 mmol/L) was aliquoted and stored at −20 °C. The rabbit polyclonal anti-CEP55 antibody was obtained from Life Technologies (Darmstadt, Germany). The rabbit polyclonal anti-SPARC antibody was obtained from Cell Signaling Technologies (Frankfurt/M., Germany). The mouse monoclonal anti-claudin-6 antibody was obtained from Santa Cruz Biotechnology (Heidelberg, Germany). For normalization of the Western blot signals, β-Actin-Ab #A5441 (obtained from Sigma Aldrich, St. Louis, MO, USA) was used at a dilution of 1:10,000. Horseradish Peroxidase-linked secondary antibodies (dilution of 1:6000) and enhanced luminescence reagents were obtained from Biozym, Oldendorf, Germany. An anti-rabbit Alexa Fluor 647-linked secondary antibody (111-605-003) was obtained from Jackson ImmunoResearch, Ely, United Kingdom, and used at a dilution of 1:500. All other chemicals were purchased from Carl Roth (Karlsruhe, Germany).