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).