Transfection of INA-6 myeloma cells and selective targeting of ERK1, ERK2, and STAT3 with siRNAs. (A) FACS analysis of INA-6 cells after electroporation with a mixture of expression vectors for EGFP, truncated CD4 (CD4 Δ), and pSUPER. From left to right: 20 hours after transfection but before purification, 24 hours after transfection and after purification of live transfected cells, and 4 days after transfection with maintenance in culture. Of note are high numbers of initially transfected myeloma cells, the selective enrichment of the most strongly transfected live cells, and the generally good viability of these cells in culture. Numbers in the inset represent the percentage of events within the respective quadrant. (B) Western blot analysis of ERK1,2 and phospho-ERK1,2 in purified INA-6 cells 72 hours after electroporation with expression plasmids for siRNAs against ERK1 or ERK2 (pSU-ERK1, pSU-ERK2), or a combination of both (pSU-ERK1 + 2). Shown is the strong and selective knockdown of the targeted protein(s), and the near absence of the respective activated form(s) even after stimulation with PMA (25 μg/mL) for 30 minutes. Basal phosphorylation of ERK became undetectable after expression of the siRNAs. (C) Western blot analysis of STAT3 in INA-6 cells electroporated with an expression plasmid for an siRNA against STAT3 (lane 2), and of cells additionally transfected with an expression plasmid (pS3R) for HA-tagged STAT3 protein derived from a sequence not recognized by the siRNA (lane 4). Shown is the near absence of STAT3 in the siRNA-transfected cells, and replenishment of the STAT3 pool through concomitant expression of the tagged, genetically modified version. Antibody staining of α-tubulin served as loading control.