Imagenflourchem fc2

Cells were washed twice with PBS and lysed with RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40) containing protease inhibitor cocktail (Roche, Mannheim, Germany) and Phenylmethylsulfonylfluoride (PMSF). Cells were afterwards sonicated (Bandelin SONOPULS) and mixed with Laemmli buffer containing 5% β-mercaptoethanol. After denaturation at 95 °C, they were run through a 15% SDS PAGE mini gel and blotted overnight using a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad, Hercules, CA, USA). Subsequently, the membrane was blocked in 5% non-fat dry milk (Carl Roth, Karlsruhe, Germany) and probed with the primary MPS1 polyclonal antibody (Table S2) followed by a secondary antibody (Table S2). β-actin served as endogenous control (Table S2). Detection was carried out using an electrochemiluminescence solution [53 (link)] and viewed with ImagenFlourChem FC2 (Cell Biosciences, Heidelberg, Australia) with the AlphaView Software (Version 1.3.0.7, ProteinSimple, San Jose, CA, USA). Densitometric analysis was carried out using Image J (NIH, Bethesda, MD, USA).

Cells were washed twice with cold phosphate-buffered saline (PBS) and lysed with RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40) containing protease inhibitor cocktail (Roche) and Phenylmethylsulfonylfluoride (PMSF). Next, cells were sonicated (Bandelin SONOPULS) and mixed with Laemmli buffer containing 5% β-mercaptoethanol. After denaturation, they were run through a 12% SDS PAGE mini gel and blotted overnight using a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad). Subsequently, the membrane was blocked in 5% non-fat dry milk (Carl Roth) and probed with the primary claudin-5 (1:500, Invitrogen), occludin (1:100, Acris), and β-actin (1: 25,000, Sigma–Aldrich), followed by secondary antibodies anti-mouse/rabbit (1:3,000, Roche Lumi Light Plus and Cell Signaling Technology) and anti-guinea pig (1:5,000, Santa Cruz). Detection was carried out using an electrochemiluminescence solution (Whitehead et al., 1979 (link)) and viewed with Imagen Flour Chem FC2 (Cell Biosciences) with the AlphaView Software (Version 1.3.0.7, Innovatech Corporation). Densitometric analyses were performed using ImageJ software (NIH and LOCI, University of Wisconsin).