Figure 1

PTEN-L is expressed in the mouse brain and in primary neuronal cell cultures. (a) Bands higher than 57 kD were observed, when primary cortical culture lysate or adult brain lysate derived from 8 week old C57BL/6 mice were blotted against PTEN. (b) Primary neuronal cultures from conditional PTEN knockout mice were transduced with Cre delivering lentiviral particles. Immunoblotting against PTEN at different time points after Cre transduction showed that PTEN protein isoforms around 57 kD and higher bands around 70–75 kD gradually decreased between day 6 and day 9 in culture, indicating genomic ablation of PTEN and PTEN-L (quantification based on n = 3 independent experiments. Values normalised against total PTEN intensity on each blot). Uncropped immunoblots are available under https://doi.org/10.6084/m9.figshare.7472984. (c) To confirm the identity of PTEN positive bands with a higher molecular weight than 57 kD, PTEN was purified using HiTrap heparin columns and subsequent immunoprecipitation from primary cortical cultures or adult cortex derived from 8 week old C57BL/6 mice. Gel pieces were extracted from silver stained gels for proteomics analysis. 500 µg of total protein was used as an input for each purification and 20% of the eluated protein fraction was loaded on the gel. (d) Six peptides were identified in mouse cortical neurons # and three peptides in adult brain lysate + that matched to the amino acid (aa) sequence unique to PTEN-L (aa 1–173) by LC-ESI-MS analysis.