Figure 2.

Construction and verification of the Kalirin Exon 13 knockout (KalSR^KO/KO). A. Diagram of the mouse Kalrn gene with exons indicated as vertical bars; Exon 13 is shown in red. B. Detail of the knockout with PCR of DNA verifying the conditional and total knockouts. C. Kalirin proteins are absent from KalSR^KO/KO cortex. Kalirin was visualized using a Kalirin spectrin antibody; the band indicated by * was not detected by Kal12 antisera and is therefore considered non-specific. The band indicated by # is thought to represent an aberrant, N-terminally shortened product of the Δ-promoter. D. PSDs prepared from WT and KalSR^KO/KO cortex were analyzed using Kal7-specific antibody; the product that appears in the KalSR^KO/KO PSDs is thought to represent the protein encoded by Exon 13-lacking transcripts initiated in the Δ-promoter. E. Subcellular fractions probed using the Kal12-specific antibody showed the presence of Kalirin12 in the supernatant of a homogenate and in the PSD fraction in WT, but not KO, extracts. Tubulin was used as a loading control. F. Quantitative PCR was used to demonstrate the absence of transcripts containing Exon 13 in the KalSR^KO mouse, while transcripts containing the Duet start were still detected.