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Figure 1. Protein results of subclones with modulated gene activity. A) Immunocytochemical analysis of Granta-519 subclones and Jeko-1 (positive control, overexpression of CDKN2A/B) for restored CDKN2-protein expression. Cells were stained for specific CDKN2B expression (green) and for nuclear DNA-content (DAPI, blue). No CDKN2B protein can be detected in original Granta-519 cells, whereas clear nuclear CDKN2B staining is observed in Granta-519 cells stably transfected with the CDKN2A/B-specific BAC-clone (+BAC-CDKN2). Jeko-1 cells overexpressing CDKN2B serve as a positive control. B) Western blot results of selected Granta-519 subclones. The subclone stably modified with pSUPERdL CCND1-specific short-hairpin insert (+shRNA-CCND1) displayed significant and specific CCND1 protein knock-down. Protein levels remained unchanged in unmodified Granta-519 clones, subclones stably modified with the empty pSUPER vector (+empty vector), with the selection cassette (+selection cassette) and with a unspecific pSUPERdL short-hairpin insert (+shRNA-unspec.). ACTB served as loading control.
