Journal: Translational Oncology
Article Title: Investigation of PP2A and Its Endogenous Inhibitors in Neuroblastoma Cell Survival and Tumor Growth
Figure Lengend Snippet: CIP2A, I2PP2A, and PP2A in neuroblastoma cell lines. (A) Immunoblotting revealed CIP2A, I2PP2A, and PP2A expression in all four neuroblastoma cell lines studied. There were no differences in expression between the MYCN nonamplified SH-EP and the isogenic MYCN amplified WAC2 cells. (B) SK-N-AS and SH-EP neuroblastoma cells ( MYCN nonamplified) were transfected with MYCN overexpression vector and cell lysates examined for I2PP2A and PP2A. MYCN was successfully expressed in both cell lines. Expression of I2PP2A and PP2A was not affected by MYCN overexpression. (C). Neuroblastoma cell lines were treated with siRNA knockdown of I2PP2A. Whole cell lysates revealed knockdown of I2PP2A with no change in PP2A expression. (D) Neuroblastoma cell lines were treated with siRNA knockdown of CIP2A. Whole cell lysates revealed knockdown of CIP2A with no change in PP2A expression. (E) PP2A activity was measured in SK-N-AS cells following siRNA inhibition. Inhibition of I2PP2A, CIP2A, or dual inhibition led to significant increases in PP2A activity.
Article Snippet: Primary antibodies used for Western blotting included the following: anti-I2PP2A (H-120) (sc-25564) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-PP2A (ab32104) and anti-CIP2A (ab99518) from Abcam (Cambridge, MA), anti–total AKT (9272), anti–phospho-AKT (S473; 9271), anti-MYCN (9405), p44/42 MAP Kinase [ERK1/2 (9102)], anti–phospho-p44/42 MAPK [phospho-ERK, T202/T204, (4377)] from Cell Signaling Technology (Danvers, MA), and anti–β-actin from Sigma (A1978, Sigma Aldrich, St. Louis, MO).
Techniques: Expressing, Amplification, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Inhibition