Figure 1.

Characterization of phospho-T840 antibody. A, Sequence alignment for amino acids surrounding S831, T840, and S845 in GluR1 across several different vertebrate species. B, Proteins in homogenates prepared from mouse hippocampi were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with the anti-phospho-T840 GluR1 antibody (lanes 2 and 3). Four distinct immunoreactive bands were detected in blots probed with the anti-phospho-T840 GluR1 antibody (antibody concentration, 0.12 μg/ml; lane 2), whereas no signal was detected in blots in which the peptide antigen used to generated the phospho-T840 GluR1 antibody was present during the primary antibody incubation (peptide concentration, 0.3 μg/ml; lane 3). Lane 1 shows biotinylated molecular weight markers, whereas lane 4 shows the same blot as lane 3 after reprobing with anti-total GluR1 antibody. C, Top, Immunoblots show bands detected by phospho-T840 GluR1 antibody before and after immunoprecipitating GluR1 from hippocampal extracts. Bottom, Immunoblots showing the effects of treating blots with λ phosphatase (2000 U for 40 min at 32°C) before incubation in anti-phospho-T840 GluR1 antibody (left set of blots). These blots were subsequently stripped and reprobed with a total GluR1 antibody (right set of blots). The arrowheads indicate the ≈ 110kD a band corresponding to GluR1. D, Western blot analysis of antibody specificity for T840 phosphorylated GluR1. GluR1 was immunoprecipitated from lysates prepared from HEK cells expressing wild-type (WT) GluR1 or a mutant form GluR1 in which T840 was changed to an alanine (T840A GluR1). Blots were probed with anti-phospho-T840 GluR1 (top), and then stripped and reprobed with total GluR1 antibodies (bottom). The first lane shows nontransfected (NT) controls.