To compensate for the partial EGFR restoration by chloroquine, the amount of EGFR precipitate loaded in the shRNA lanes was increased by 25%%.
The dialyzed ammonium sulphate precipitate was loaded on a manually packed DEAE sepharose column equilibrated with 50 mM Tris-Cl (pH 8.0) containing 0.18 M NaCl.
One quarter of the TCA precipitate was loaded on a 10% SDS-PAGE gel.
Total of 5 mL of ammonia sulfate precipitate was loaded onto a Sephadex G-100 column (1.5 × 65 cm).
The 40 80% (NH4 2SO4 precipitate was loaded onto a chromatographic column (14 × 25 cm, 3,800 mL) of DEAE-Sephadex A25 (Amersham Biosciences, Uppsala, Sweden) previously equilibrated on 10 mM Tris-HCl buffer (pH 7.6).
Insoluble material was removed via centrifugation at 35000 g for 30 min and the supernatant cleared by filtration (Whatman No. 1) to remove residual precipitate, before loading onto a 1 ml HisTrap column (GE Healthcare), equilibrated with EQ buffer at ≤0.5 ml/min.
Samples were then TCA precipitated and loaded on 5 20% gradient gels and either silver stained or western blotted.
Reactions products were precipitated and loaded on the DNA sequencer as directed by the manufacturer (Beckman Coulter Model CEQ8000).
Reactions were treated with exonuclease III and terminated as described above, following which they were phenol extracted and ethanol precipitated and loaded on a prerun 8% polyacrylamide gel and electrophoresed using 0.5% TBE running buffer.
Following incubation, samples were treated with proteinase K extraction buffer (200 mM Tris HCl pH 7.5, 25 mM EDTA pH 8.0, 300 mM NaCl, 2% SDS, 0.3 μg/μl proteinase K) at 65°C for 20 min; RNA was phenol chloroform extracted, precipitated and loaded on 10% PAGE.
