MATERIALS AND METHODS
Materials
Antibodies were from Cell Signaling Technology (GAPDH, PTEN, phospho-PTEN
Ser380/Thr382/383, Shp1), Abcam (Shp2), and Bethyl Laboratories (PHLPP). The
following Taqman probes were used for qPCR analysis: PTEN (Mm00477208_m1),
PHLPP (Mm01295850_m1), Shp1/PTPN6 (Mm00469153_m1), Shp2/PTPN11
(Mm00448434_m1),
Ship-1/Inpp5d
(Mm00494987_m1),
Ship-2/Inppl1
(Mm00802853_m1), and GAPDH (Mm99999915_g1).
Animals
All animal experiments were performed in compliance with approved policies of the
University of Colorado School of Medicine. Mice expressing constitutively active Akt
in PLP-expressing cells[37] and WT littermates were anesthetized with isoflurane and
the corpus callosum was microdissected, flash frozen in liquid nitrogen, and stored at
-80°C.
Western Blot
Corpus callosum protein lysates were prepared by homogenization in RIPA buffer
supplemented with protease inhibitor tablet (Roche) and phosphatase inhibitor
cocktail (CalBiochem). Twenty micrograms of protein was separated (4-20%
SDS-PAGE, Bio-Rad) and transferred to a polyvinylidene fluoride (PVDF)
membrane. Membranes were blocked in 5% BSA in TBS and incubated with the
following primary antibodies: PTEN, 1:1000; phospho-PTEN, 1:1000; PHLPP,
1:2500; GAPDH, 1:5000; Shp2, 1:1000; Shp1, 1:1000. Membranes were washed,
incubated with the appropriate secondary antibody (LI-COR), and imaged/quantified
using an Odyssey infrared imaging system (LI-COR). GAPDH was used as a loading
control. Three animals per group were used for protein expression analysis. Group
means were normalized to P7 WT.
Quantitative Real-Time Polymerase Chain Reaction (qPCR)
RNA extraction and cDNA synthesis were performed as previously described[148]. All
qPCR experiments were performed using Taqman probes on a StepOnePlus real-time
PCR system (Applied Biosciences), according to the manufacturer’s instructions. At
least three animals per group were used for gene expression analysis and each sample
was run in triplicate. The delta(delta(Ct)) method was used to assess expression
changes, using GAPDH as an internal control. All samples were run simultaneously
and the gene expression for each sample was normalized to the average of three P7
WT samples.
Statistical Analysis
All statistical analyses were performed using unpaired Student’s t-tests. Statistical
significance was set at P <0.05.