Validation and functional testing of novel isoforms.

A, Expression of 15 isoforms was assessed by qPCR in DRG neurons and CGNs. Isoform expression was compared to the fold change in FPKM. (Relative expression and FPKM ratios in Table 3). B, C, Atf3 and Pten isoform specific primers (bottom panels) were used on DRG cDNA and produced PCR products of the predicted sizes. B, Lane2: Atf3 cDNA (Open BioSystems); Lane3: conventional Atf3; Lane4: Atf3 J1; Lane5: Atf2 J2; Lane 6: Atf3 J3; Lane7: no template control; Lane 8: no reverse transcription (RT) control. C, Lane2: conventional Pten; Lane3: no template control for Pten; Lane4: no RT control for Pten; Lane5: Pten J2; Lane 6: no template control for Pten J2; Lane 7: no RT control for Pten J2. Schematic representations of Atf3 and Pten isoforms are below each gel (not to scale). Primer positions are indicated with colored arrows. D, The ratio of Pten J2 expression to Pten conventional + Pten J1 expression (in FPKM). The FPKMs for Pten and Pten J1 were summed because there is no way to distinguish the isoforms by PCR. Pten J2 expression is reduced 80–90%. E, qPCR validates the reduction in Pten J2 expression. F, Western blot for PTEN (50 kD) and PTEN J2 (32 kD) confirms that both proteins can be produced from the corresponding cDNAs.