Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

Figure 2

RAG1 protein expression in amygdalar neuronal cells. Amygdalar coronal sections of context fear conditioning-trained mice, perfused 1 h after conditioning, were used for immunofluorescence and analyzed by confocal microscopy. Antibodies from immunofluorescence were validated by Western blot analysis. (a) Amygdalar area representative images of a double immunostaining using RAG1 antibody labeled with Alexa Fluor 488, green channel signal, and NeuN antibody labeled with Alexa Fluor 568, red channel signal. The left panel shows the NeuN positive neuronal nuclei, while the middle panel depicts RAG1 immunopositive cells. The right panel is the merge image showing colocalization of the NeuN neuronal nuclei marker and RAG1. Arrows point to some of the RAG1 immunopositive neurons. These immunofluorescent images revealed colocalization of RAG1 protein expressing cells with those expressing NeuN, suggesting the presence of RAG1 in neurons, although not all neurons expressed RAG1. (b) Tissue punches from amygdala (Amy) were obtained 1 h after context fear conditioning and analyzed in Western blot by comparative comigration with a standard molecular weight (MW) marker and protein extracts from bone marrow (BM) ((b)-1) and thymus (Thy) ((b)-2). Both sets of experiments consistently showed comigration between the tissues with a band corresponding to ~120 KD of RAG1 protein (green channel corresponding to RAG1 and red channel corresponding to beta-actin, ~42 KD); prestained molecular weight (MW) marker (ladder) was included in all the Western blots. ((b)-3) Additionally, tissue protein extracts from leg muscle (Mus) (negative control) were analyzed compared to amygdalar extracts with respect to RAG1 expression. As expected, RAG1 was not expressed in muscle compared to amygdala ((b)-3), bone marrow ((b)-1), and thymus ((b)-2). ((b)-4) RAG1 antibody preabsorption assays, either with muscle or with bone marrow extracts, showed that only bone marrow extracts, which express RAG1 as opposed to muscle, were able to block the ~120 KD band from amygdalar protein extracts in the Western blots, indicating that RAG1 antibody was preabsorbed (blocked) only by RAG1 protein expressing tissue (bone marrow).

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