Anti mid51

For immunohistochemistry analyses cells were seeded on x-well Tissue Culture Chambers (Sarstedt, Nümbrecht, Germany) and transfected for 48 h. Cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.2% Tween20 for 5 min in phosphate-buffered saline. Cells were stained for 1 h with the primary antibodies: anti-MiD51 (1:100) (Proteintech), anti-Tom20 (1:100) (Abcam, Cambridge, UK), anti-insulin (1:100) (Abcam) and anti-LC3 (1:100) (Sigma). Cy5 or FITC-coupled secondary antibodies (1:250) were used for visualization (Molecular Probes Invitrogen, Darmstadt, Germany). Cells were mounted and counterstained using Roti^®-Mount FluorCare DAPI (Roth, Karlsruhe, Germany) and analyzed using a Fluoview FV10i confocal microscope (Olympus, Hamburg, Germany).

Cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (50 mmol/l Tris-HCl pH 7.4, 150 mmol/l NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/l EDTA, protease inhibitors) and centrifuged for 15 min at 12.000 g. 30 μg protein were separated by SDS-PAGE and blotted onto Roti^®Fluoro PVDF membrane (Roth, Karlsruhe, Germany). Membranes were incubated for 1 h at room temperature with the following primary antibodies: anti-c-Myc (1:1,000) and anti-GAPDH (1:1,000) (Santa-Cruz Biotechnology, Santa Cruz, CA, USA) or anti-MiD51 (1:1,000) (Proteintech, Rosemont, IL, USA) and anti-beta-actin (1:200) (Santa Cruz). Immunoreactive bands were visualized using the following fluorescence-labeled secondary antibodies: IRDye 680 CW, IRDye 800 CW and analyzed via the Odyssey imaging system. Densitometry measurements of bands were performed using the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA).