Data Set 3: Proximity ligation assay of PTEN-L:EGFP (V420) with Gab2 after control (PBS) or 50µM glutamate treatment (Fig. 4c)
This dataset contains 18 .tif image files holding the results of a proximity ligation assay of PTEN-L:EGFP (V420) with Gab2 after control (PBS) or 50µM glutamate treatment. These images correspond to figure 4 of the related article: Unique properties of PTEN-L contribute to neuroprotection in response to ischemic-like stress.
The related study investigates the function and regulation of PTEN-L in neurons in the context of brain ischemia. Exogenous, EGFP-tagged PTEN-L or the shorter variant PTEN were expressed in primary neurons after knock out of endogenous PTEN. Oxygen-glucose deprivation or exposure to 50 µM glutamate were applied to neurons to test their response to ischemic-like stress.
Figure 4 | Unique interaction of PTEN-L:EGFP in response to glutamate stress and cytosolic interaction with Gab2.
A proximity-ligation assay with a Gab2 and PTEN-L antibody was performed in PTEN-L replaced neurons. These image data present the interaction between Gab2 and PTEN-L under PBS conditions, including changes after 50μM glutamate was applied, as described in the related research article.
Primary neurons derived from conditional PTEN knockout mice were plated in 8-well microscopy dishes (Ibidi) and transduced with either cre- and PTEN-delivering lentiviral particles LVPs (Knockout and exogenous PTEN replacement condition), or mcherry- and EGFP-delivering control LVPs (Endogenous PTEN condition). At DIV 9 cells were treated with either PBS or 50 µM glutamate for 60 min and fixed with 4 % formaldehyde. To test the primary antibodies anti-GAB2 (HPA001368, Sigma-Aldrich), derived from murine origin, and anti-PTEN-L (MABS1680, Merck), derived from canine origin, cells were permeabilised with 0.25% triton X-100 , blocked with 0.2% normal donkey serum (ab7475, Abcam) and primary antibodies were applied at 1:500 dilutions over-night. Secondary antibodies alexa Fluor 647 donkey anti-rabbit IgG and alexa Fluor 647 donkey anti-mouse IgG (ThermoFisher) were applied at 1:500 dilutions for 1 h at room temperature.
To detect interactions between GAB2 and PTEN-L below a distance of 40 nm, permeabilisation, blocking and primary antibodies were applied like described above. Duolink® PLA PLUS targeting mouse IgGs and Duolink® PLA MINUS secondary antibodies targeting rabbit IgGs were incubated at ambient temperature overnight. Ligation, rolling circle amplification and hybridization with in the far red detectable olgionucleotides were performed according to the manufacturer’s instructions (Duolink ® PLA, Sigma Aldrich).