Cell lysis, gel electrophoresis and Western blotting
Cells were cultured in 24-well-plates. Lysates were fractioned into a soluble cytosolic and insoluble cytoskeletal bound fraction using triton-extraction-buffer (0.5 % Triton X-100, 50 mmol/l MES, 25 mmol/l EGTA, 5 mmol/l MgCl2, pH 6.8, 0.1 % pepstatin+aprotinin+leupeptin, 1 % PMSF) for 10 min on ice under gentle shaking. The pellet=cytoskeletal fraction was separated at 14.000 rpm for 10 min at 4° C and the supernatant=cytosolic fraction was retrieved. The pellet was washed 1x and lysed with ultrasound in SDS lysis buffer (25 mM HEPES, 2 mM EDTA, 25 mM NaF, 1 % SDS, pH 7.6, cOmplete™ (Merk, USA)). Protein amount was determined with a commercial Pierce BCA protein assay kit. Western-blotting was performed, using a standard wet blotting protocol on nitrocellulose membranes (Life Technologies, USA). Membranes were blocked with ROTI®Block (Carl Roth, Germany) 1:10 in Tris-buffered saline with 0.05 % tween (TBST) for 1 h antibodies were used overnight at 4 °C in 5 % BSA in TBST 1:1000, except anti-p-PKC α (1:20.000). Anti-rabbit/mouse horseradish-peroxidase-coupled secondary antibodies (Dianova, Germany) were used 1:10.000 in TBST for 1 h and visualized with self-made ECL solution on a FluorchemE developer (Protein Simple, USA).