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Figure 1 Comparison of C-terminal amino acid sequences of rat Fnk and Snk. Amino acid residues of Fnk (393–556) and Snk (412–590) are aligned. Identical amino acids are highlighted in blue. The polo-box (Polo30) and a larger region (Polo70) used in the two-hybrid analyses are framed and shown in dark and light gray, respectively. The proteins share 76.6% sequence identity in the polo-box region (Polo30).
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 | Figure 2 Regulation of Fnk and Snk mRNA levels in the hippocampus. (A) Autoradiograph of Northern blot analysis of Fnk-specific transcripts. A 2  g aliquot of poly(A)+ RNA was loaded per lane. The blot was hybridized to a probe specific for Fnk. Hybridization to a probe specific for GAPDH was used as a loading control. Lane C, mRNA from saline-injected animals. Lane 1, mRNA isolated 1 h after PTZ-induced seizures. (B) Quantification of Fnk Northern blots given in bar diagrams. Error bars indicate SEMs (n = 3). Abbreviations are as in (A). (C) Autoradiograph of Northern blot analysis of Snk-specific transcripts. A 5 g aliquot of total hippocampal RNA was loaded per lane. The blot was hybridized to probes specific for Snk and GAPDH. Lane C, RNA from saline-injected animals. Lanes 1, 4 and 10, the numbers indicate the time in hours after the onset of PTZ-induced seizures. Lane C/P, RNA isolated 4 h after the onset of PTZ-induced seizures in the presence of CHX. Lane K4, RNA isolated 4 h after the onset of KA-induced seizures.
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Figure 3 Comparative analysis of brain Fnk and Snk mRNA levels before and after seizure. Coronal sections were analyzed for Fnk mRNA (A, C and E) and Snk mRNA (B, D and F) using in situ hybridization with gene-specific antisense probes. (A and B) Control rat; (C and D) rat sacrificed 4 h after PTZ-induced seizure; (E and F) rat sacrificed 4 h after KA-induced seizures. a, amygdala; c, cortex; CA1–3, fields CA1–3 of the hippocampus; dg, dentate gyrus; mhb, medial habenula; sth, subthalamic nucleus; VI, layer VI of the cortex.
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 | Figure 4 Induction of LTP induces Fnk and Snk mRNAs in dentate gyrus granule cells in freely moving rats. Coronal sections were assayed for Fnk and Snk mRNA using in situ hybridization with gene-specific antisense probes. (A and B) Superimposed field potentials before and 1 h after (A) low-frequency stimulation (LFS) and (B) high-frequency stimulation (HFS) showing the induction of LTP with the latter. (C and D) Fnk mRNA levels 1 h after unilateral application of (C) LFS or (D) HFS. (E and F) Snk mRNA levels 1 h after unilateral application of (E) LFS or (F) HFS. The scale bar in (A) and (B) is 5 mV/2 ms.
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Figure 5 Fnk and Snk proteins are localized to neuronal somata and dendrites of stimulated hippocampal neurons. Sections through hippocampi of control and KA-stimulated rat brain (4 h survival) were analyzed for Fnk protein (A, C, E, F, I and J) or Snk protein (B, D, G, H, K and L) using affinity-purified antisera. (A and B) Control hippocampus, only weak Fnk (A) and Snk (B) immunoreactivity is detected within the dentate gyrus and fields CA1–3. (C and D) After KA-induced seizures, Fnk (C) and Snk (D) immunoreactivity is increased in the granular and molecular layer of the dentate gyrus and in region CA1 with prominent staining of the dendritic processes. (E–H) High-power views of granule cells of the dentate gyrus before (E and G) and after KA-induced seizures (F and H). (I–L) High-power views of hippocampal field CA1 before (I and K) and after (J and L) KA-induced seizures. Sections from the same animal incubated with a serum depleted of either Fnk (M) or Snk (N) antibodies had no staining. (O) Immunoblots demonstrating the specific binding of the antisera to the corresponding recombinant kinase protein and that no cross-reactivity was observed. Recombinant Fnk protein (100 ng) was reacted with immunodepleted Fnk-specific antisera (lane 1), Fnk-specific antisera (lane 2) and Snk-specific antisera (lane 6). Recombinant Snk protein (100 ng) was reacted with immunodepleted Snk-specific antisera (lane 4), Snk-specific antisera (lane 5) and Fnk-specific antisera (lane 3). CA1–3, hippocampal fields CA1–3; dg, dentate gyrus; g, granular cell layer; p, pyramidal cell layer; slm, stratum lacunosum moleculare; sm, stratum moleculare; so, stratum oriens; sr, stratum radiatum.
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 | Figure 6 Cib mRNA is expressed in rat brain. Autoradiograph of Northern blot analysis of RNA extracted from cortex (A) and in situ hybridizations of coronal sections with gene-specific sense (B) and antisense (C and D) probes for Cib. (A) A 2 g aliquot of poly(A)+ RNA was loaded. The blot was hybridized to a probe specific for Cib. Size markers in kilobases are indicated on the left. (B and C) Control rat, (D) rat sacrified 4 h after PTZ-induced seizure. CA1–3, hippocampal fields CA1–3 of the hippocampus; dg, dentate gyrus; mhb, medial habenula.
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Figure 7 Cib protein is localized to neuronal somata and dendrites of hippocampal neurons. Sections through hippocampi of rat brain were analyzed for Cib protein using a monoclonal mouse anti-Cib antibody (A–C). Immunoreactivity is detected within the dentate gyrus and fields CA1–3 of hippocampus (A). (B and C) High-power views show immunoreactivity in the granular and molecular layer of the dentate gyrus (B) and region CA1 with prominent staining of the dendritic processes (C). CA1–3, hippocampal fields CA1–3; dg, dentate gyrus; g, granular cell layer; p, pyramidal cell layer; slm, stratum lacunosum moleculare; sm, stratum moleculare; so, stratum oriens; sr, stratum radiatum.
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 | Figure 8 Analysis of interactions between Snk and Cib, and Fnk and Cib. (A) Yeast two-hybrid interaction analysis for Snk and Cib. (B) Yeast two-hybrid interaction analysis for Fnk and Cib. Assays in (A) and (B) were performed in liquid culture and  -galactosidase activity was quantified. The enzymatic assays shown represent the average of three independent co-transformants. Interaction was only observed between Cib and either the full-length kinase proteins, C-terminal fragments or polo-box domains. No interaction was seen between full-length kinases and lamin C, between Cib and an N-terminal Snk fragment, and between Cib and trypsinogen which was used as an additional negative control. Snf4 and Snf1 served as positive controls. Cib, complete coding region of Cib; Fnk, complete coding region of Fnk; FnkCT, C-terminal 313 amino acids of Fnk; FnkPolo30, 30 amino acids of the polo-box of Fnk; FnkPolo70, 70 amino acids of the polo-box domain of Fnk; Snk, complete coding region of Snk; SnkCT, C-terminal 331 amino acids of Snk; SnkNT, N-terminal 352 amino acids of Snk; SnkPolo30, 30 amino acids of the polo-box of Snk; SnkPolo70, 70 amino acids of the polo-box domain of Snk. (C) In vitro binding analysis of the interaction between Snk and Cib, and between Fnk and Cib. Myc-tagged GST protein (GST), Myc-tagged Snk C-terminal fusion protein (GSTSnkCT) and Myc-tagged Fnk C-terminal fusion protein (GSTFnkCT) were bound to HA-tagged GST–Cib fusion protein. The amount of bound Cib was quantified using a monoclonal mouse anti-HA antibody and a secondary peroxidase-conjugated mouse antibody. Relative binding data were: GST, 100%; GSTFnkCT, 925%; GSTSnkCT, 1114%.
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Figure 9 Snk and Cib show identical subcellular localization when expressed together. Optical sections of single- and double-transfected COS and N2A cells were analyzed using confocal microscopy for the expression of GFP–Snk (green) or HA-tagged Cib detected by a Cy3-labeled secondary antibody (red). (A) Single transfected COS cell expressing GFP–Snk fusion protein. (B) Single transfected COS cell expressing HA-tagged Cib fusion protein. (C) Single transfected COS cell expressing HA-tagged Cib fusion protein with exclusive nuclear localization. (D) Double transfected COS cell expressing GFP–Snk fusion protein and HA-tagged Cib fusion protein. The localization of GFP–Snk fusion protein is shown. (E) The same cell as in (D). The localization of Cib fusion protein is shown. (F) Superposition of images shown in (D) and (E). (G) Single transfected N2A cell expressing GFP–Snk fusion protein. (H) Single transfected N2A cell expressing HA-tagged Cib fusion protein. (I) Phase-contrast image of the field containing the Cib-expressing cell shown in (H). In the field shown, there are several non-Cib-expressing cells which have no background staining as can be seen in (H). (J) Double transfected N2A cell expressing GFP–Snk fusion protein and HA-tagged Cib fusion protein. The localization of GFP–Snk fusion protein is shown. (K) Same cell as in (J). The localization of Cib fusion protein is shown. (L) Superposition of images shown in (J) and (K).
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 | Figure 10 Snk and Cib proteins co-localize in the somata and dendrites of hippocampal neurons. Sections through hippocampal region CA1 of control and KA-stimulated rat brain (4 h survival) were analyzed using confocal microscopy for the expression of Snk protein (Cy2, green) (A and D) and Cib protein (Cy3, red) (B and E). Images were superimposed in (C) and (F). (A and B) Control hippocampus; Snk (A) and Cib (B) immunoreactivity is detected in the subplasmalemmal cortex of the neuronal cytoplasm and dendritic processes. (D and E) After KA-induced seizures, immunoreactivity of Snk (D) is increased whereas that of Cib remains unchanged (E). (C) Superposition of images shown in (A) and (B), and (F) images shown in (D) and (E), demonstrates extensive co-localization of Cib and Snk in the cytoplasm and dendritic processes.
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