Upon thawing and re-suspension in 100 mM potassium phosphate buffer with pH 7.5 (5 ml g−1 wet cell weight), lysozyme (final conc. 1 mg ml−1) and a protease inhibitor mix (Complete Protease Inhibitor Cocktail Tablets, Roche) were added.
In turn, the monolayers were resuspended in 40 µl of lysis buffer (1% Triton X-100) and protease inhibitor mix (Complete™ protease inhibitors, Roche).
Yeast cells were grown to OD600 of 0.8 1.2, harvested and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris.Cl, 5 mM EDTA, 1% Triton X-100, pH 7.5) plus protease inhibitor mix (Roche, Indianapolis).
Cells were lysed in buffer containing 20 mM Hepes, pH 7.5, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol and a protease inhibitor mix (Roche, Basel, Switzerland).
After three washes with 1 ml of washing buffer (lysis buffer without protease inhibitor mix), bound material was eluted under denaturing conditions and separated on SDS-PAGE gels and transferred to PVDF membranes.
The HEK cells were then lysed for 20 min at 4°C in lysis buffer (150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 50 mM Tris-HCl (pH 7.5)) and a protease inhibitor mix (Pierce Biotechnology).
Cells were then lysed using 20 mM HEPES pH 7.3, 1% Triton X-100, 100 mM NaCl, 20 mM MgCl2, 1 mM Dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and complete protease inhibitor mix (Roche).
For co-immunoprecipitation, cells were lysed in buffer containing 25 mmol/L Tris (pH 7.5), 225 mmol/L NaCl, 1% Triton X-100, 10% glycerol, phosphatase inhibitors (10 mmol/L NaF, 1 mmol/L Na2VO4), and protease inhibitor mix (Roche).
For Western blots mouse livers (100 mg) were homogenized in 1× NuPAGE LDS loading buffer (cat. #NP0007, Invitrogen, Carlsbad, CA) with 1× protease inhibitor mix (cat. #8340, Sigma-Aldrich, St . Louis MO), 1× Phosphatase Inhibitor Cocktail Set 1 (cat. #524624, Calbiochem, San Diego, CA) and 1× Phosphatase Inhibitor Cocktail Set 2 (cat. #524625, Calbiochem, San Diego, CA).
Cells were harvested 48 h after transfection with a sterile cell scraper (Nunc) in 1.5 ml of ice-cold IP-lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Igepal and EDTA-free inhibitor mix: Roche Applied Science).
For cell fractionation 100 mg tissue of transiently transformed tobacco leafs were homogenised in liquid nitrogen and the homogenate was extracted in 2 ml homogenization buffer (25 mM MOPS, 0.1 mM MgCl2, 8 mM L-cysteine, 2.5 mM EDTA, 2× protease inhibitor mix (Roche), 250 mM sucrose; pH 7.8).
