Subcellular fractionation and immunoblotting of WT and CS
hippocampal or forebrain (cortex and hippocampus) lysates were performed as
in (Sanderson et al., 2016 (link); 2012 (link); Smith et al., 2006 (link);Grosshans et
al., 2002 ). For immunoblotting, 15 μg of whole extract
(WE), 10 μg of P2, 20 μg of S2, 5 μg of TxP, and 15
μg of TxS were resolved on Tris-SDS gels and transferred in 20%
methanol to PVDF membranes. Membranes were incubated with primary antibodies
for 2 hr as follows: rabbit anti-AKAP150 (1:1000) (Brandao et al., 2012 (link)), mouse anti-PKA-RIIβ
(1:1000; BD Biosciences Transduction Laboratories), mouse anti-PSD-95
(1:1000; Millipore), rabbit anti-GluA1 (1:1000; Millipore), and rabbit
anti-GluA1-S845 (1:1000; Millipore). Signal detection was performed using
HRP-coupled secondary antibodies (Bio-Rad; 1:10,000) followed by ECL (West
Pico or West Dura Chemiluminescent Substrate; Pierce). Chemiluminescence was
imaged using an Alpha Innotech Fluorchem gel documentation system, and band
intensities were analyzed using ImageJ (NIH). Band intensities were
normalized to WT WE from the same blot.
hippocampal or forebrain (cortex and hippocampus) lysates were performed as
in (Sanderson et al., 2016 (link); 2012 (link); Smith et al., 2006 (link);
al., 2002
(WE), 10 μg of P2, 20 μg of S2, 5 μg of TxP, and 15
μg of TxS were resolved on Tris-SDS gels and transferred in 20%
methanol to PVDF membranes. Membranes were incubated with primary antibodies
for 2 hr as follows: rabbit anti-AKAP150 (1:1000) (Brandao et al., 2012 (link)), mouse anti-PKA-RIIβ
(1:1000; BD Biosciences Transduction Laboratories), mouse anti-PSD-95
(1:1000; Millipore), rabbit anti-GluA1 (1:1000; Millipore), and rabbit
anti-GluA1-S845 (1:1000; Millipore). Signal detection was performed using
HRP-coupled secondary antibodies (Bio-Rad; 1:10,000) followed by ECL (West
Pico or West Dura Chemiluminescent Substrate; Pierce). Chemiluminescence was
imaged using an Alpha Innotech Fluorchem gel documentation system, and band
intensities were analyzed using ImageJ (NIH). Band intensities were
normalized to WT WE from the same blot.