Fig. 2.

a Nucleotide sequence of caPeptide placed into expression vector along with the corresponding amino acid translation. The amino acids corresponding to a.a. 126–133 of caPCNA are underlined. The peptide sequence was followed by a three nuclear localization signal repeat (3 × NLS). b Diagram representation of expression vector used for creation of stable inducible cell population. Expression vector features include the tetracycline inducible promoter, pTight, caPeptide-3 × NLS followed by the co-translational cleavage moderator, 2a peptide and the fluorescent reporter gene, mcherry. Also present is a puromycin resistance selectable marker. c Bright field and fluorescence composite microscopy image showing Dox induction of caPeptide and reporter protein mcherry in 231-caPeptide cell lines. Induction results in a single transcript containing the coding information for the respective peptide and mcherry reporter which is then translated in a single ribosomal event but cleaved into peptide and reporter gene through the activity of the 2a peptide sequence. This results in equimolar expression of peptide and reporter gene. d Reverse transcription PCR of mRNA from Dox-treated and untreated 231-caPeptide cell populations. Reaction #1 used primers that annealed to the 5′ and 3′ ends of the mcherry sequence in the cDNA template. Reaction #2 used primers that annealed to the 5′ end of caPeptide and same 3′ end primer as reaction #1. The expected products are 770 and 930 bp, respectively. e Lysates of 231-v.c cells (vector control) and 231-flg-caPeptide cells metabolically labeled with tritiated lysine in the presence and absence of doxycycline were immunoprecipitated with an antibody specific to the flag tag (Anti-Flag) and an antibody developed by using the caPeptide sequence as an immunogen (Anti-caPCNA). The samples were separated by gel electrophoresis and visualized by autoradiography