Selection of ALK shRNA. (A) ALK-A5 efficiently inhibits NPM-ALK protein expression. HEK-293T cells were cotransfected with Pallino NPM-ALK (0.2 μg) and 1 of 6 different pSUPER vectors carrying shRNA specific for ALK sequences (pS-A1-6; 0.8 μg). Untransfected cells were also included (–). Lysates (72 hours) were immunoblotted using a specific anti-ALK antibody recognizing the cytoplasmic region of the ALK-R. Protein loading was normalized using anti–α-tubulin antibody. (B) shRNA ALK-A5 does not modulate the expression of TPR-MET. HEK-293T cells transfected with TRP-MET and ALK-A5 pSUPER vectors express unmodified levels of TPR-MET. However, TPR-MET expression is down-modulated after cotransfection with a specific TPR-MET (TM2) shRNA construct (top panel).37 HEK-293T cells transfected NPM-ALK and the TPR-MET shRNA cassette expressed unmodified levels of NPM-ALK (bottom panel). (C) ALK5 inhibits ATIC-ALK protein expression. HEK-293T cells were cotransfected with Pallino ATIC-ALK and ALK-pSUPER shRNA interfering sequences. Expression of ATIC-ALK protein was determined by Western blot analysis as described in “Materials and methods.” (D) ALK-A5 inhibits ALK-R protein expression. HEK-293T cells were cotransfected with Pallino ALK-R and pSUPER-ALK shRNA interfering sequences. Protein loading was normalized using a rabbit polyclonal antibody to CDK2.
