Western blue reagent

A 0.2 gram pellet from each transfected culture was lysed in 200μl cold lysis buffer with protease inhibitor cocktail. Cells were lysed on ice using a Missonix XL-2000 ultrasonic cell disruptor for 3 × 10 pulses at power 2. Total cell lysate was diluted in 5X Laemmli sample buffer. Clarified soluble fraction was generated by centrifugation at 14,000 × g for 30 minutes at 4°C and mixed with 5X Laemmli sample buffer. Samples were boiled for 5 minutes and separated electrophoretically on 4–20% SDS-PAGE gels (Bio-Rad) using a Bio-Rad mini protean system. For SDS-PAGE analysis the gel was stained with Coomassie brilliant blue stain. For Western blot analysis the proteins were transferred onto a PVDF membrane (Millipore) using a semi-dry transfer apparatus (GE Healthcare). Membrane was blocked in TBST buffer (25mM Tris-HCl pH7.5, 130mM NaCl, 0.1% Tween-20) with 5% fat-free dry milk for 30 minutes. The membrane was incubated in the indicated primary antibody overnight at 4°C. After washing the membrane 3 × 10 minutes in TBST, the membrane was incubated in alkaline phosphatase-conjugated secondary antibody for 2 hours at room temperature. The bands were visualized using Western blue reagent (Promega). Images were acquired with a Canon LiDE 200 scanner.