Figure 1.
The C-terminal 7 aa of GluR1 are required for its association with SAP97; both proteins are found in the neonatal spinal cord.
A
, HEK 293 cells were cotransfected with expression vectors for SAP97 + GFP-tagged GluR1 or GFP-tagged GluR1Δ7, and lysates were immunoprecipitated with anti-SAP97 and probed with anti-GFP, anti-GluR1, or anti-SAP97. Full-length GluR1 (detected with either anti-GFP or anti-GluR1), but not GluR1Δ7, could be pulled down with anti-SAP97.
B
, Spinal cord lysates from WT or GluR1Δ7 P7 mouse pups were immunoprecipitated with anti-SAP97 and blotted with anti-GluR1 or anti-SAP97. Endogenous full-length GluR1, but not GluR1Δ7, is pulled down with anti-SAP97. Levels of expression of GluR1, GluR1Δ7, and SAP97 are equivalent in the mice of either genotype.
C
, Spinal cord lysates from WT P7 pups were immunoprecipitated with anti-SAP97 or no primary antibody and probed with anti-SAP97, anti-GluR1, or anti-CASK. Protein complexes containing GluR1, SAP97, and CASK were pulled down only when immunoprecipitating with anti-GluR1. The level of GluR1 in the starting material (input) was equivalent.
D
, Lysates from HEK 293 cells heterologously expressing the α-isoform or β-isoform of SAP97 were run along with P7 spinal cord lysates and blotted for SAP97. Spinal cord SAP97 migrates at the same molecular weight as the β-isoform, indicating that this is the predominantly expressed endogenous species.
E
, SAP97 is present in lysates from WT mouse spinal cord at P7, P14, and P21 and mouse brain at P14 and P21. GluR1 is expressed at a lower level in the spinal cord at P7–P14 in comparison with the brain.
F
, Immunostaining of the P7 spinal cord with anti-SAP97 reveals staining of motor neurons in the ventral horn. (Photomicrograph printed with permission from Dr. Maria Rubio, University of Connecticut, Storrs, CT.) IP, Immunoprecipitation; GM, gray matter; WM, white matter.