Figure 2. Immunodetection of in vitro and in vivo expressed FKHR-PAX3 protein.

(A) Left panel: schematic of deduced sizes and amino acid sequence variations for FKHR-PAX3 isoforms c, d, and e. Right panel: Autoradiographic image of S³⁵-methionine labeled in vitro translated FKHR-PAX3 protein isoforms. (B) Verification of PAX3-specific C2 and FKHR-specific L27 antibodies in detecting in vivo expressed FKHR-PAX3 proteins. Top panel: diagrammatic illustration of the epitope locations within the FKHR-PAX3 protein recognized by L27 and C2 antibodies. Bottom panel: western blot detection of FKHR-PAX3 in whole cell extracts (30 μg) prepared from RD cells that were transiently transfected with control expression vector (lane1), FKHR-PAX3 isoform c (lane 2), and FKHR-PAX3 isoform d (lane 3) using C2 (left panel) and L27 (right panel) antibodies. (C) Western blot detection of the endogenously expressed FKHR-PAX3 in RH28 and RH30 cells by L27 and C2 antibodies. Protein extract from FKHR-PAX3 negative RH4 cells was included as negative control. n.s.: non-specific bands resulting from high amount of protein extracts used and long film exposure. (D) Effect of PAX3-FKHR knockdown on the endogenous level of FKHR-PAX3 in RH28 and RH30 cells. Whole cell extracts were prepared from cells that stably expressed the inducible PAX3-FKHR shRNA treated with DMSO or DOX for 48 hours, and analyzed for FKHR and FKHR-PAX3 expression. (E) Effect of MG132 (10 μM for 12 hours) and 5’-Aza-C (1 μM for 48 hours) on endogenous FKHR-PAX3 expression levels in RH28 and RH30 cells. (C-E) A total of 400 μg of protein extracts were used for the analyses. (D-E) Alpha-tubulin was used to normalize sample loading.