Journal: Japanese Clinical Medicine

Article Title: Dynamic Change in Cells Expressing IL-1β in Rat Hippocampus after Status Epilepticus

doi: 10.4137/jcm.s13738

Figure Lengend Snippet: Figure 2. IL-1b immunoreactivity in the hippocampus of control rat without SE and after SE. (A, D, G, J, M) Expression of IL-1b at control rat hippocampus. (A) Expression of IL-1b at the hippocampus of control rat without SE in low-power magnification. Scale bar = 1 mm. (D) CA1 region of control rat in intermediate power magnification. (G) CA3 region of control rat in intermediate power magnification. D, G; scale bar = 100 μm. (J) CA1 region of control rat in high-power magnification. (M) CA3 region of control rat in high-power magnification. J, M; scale bar = 50 μm. Expression of IL-1b was faintly observed in the pyramidal cells at CA1 (D, J) and CA3 (G, M) regions. (B, E, H, K, N) Expression of IL-1b at day 1 after SE. (B) Expression of IL-1b at the hippocampus at day 1 after SE in low power magnification. Scale bar = 1 mm. (E) CA1 region at day 1 after SE in intermediate power magnification. (H) CA3 region at day 1 after SE in intermediate power magnification. E, H; scale bar = 100 μm. (K) CA1 region at day 1 in high power magnification. (N) CA3 region at day 1 after SE in high power magnification. IL-1b expression increased transiently in the cytoplasm of the remaining pyramidal cells in the CA3 region beginning on day 1 after SE (H, N). Expression of IL-1b in the cytoplasm of pyramidal cells at CA3 was greater than that at CA1 on day 1 after SE (E, H, K, N; Table 1). K, N; scale bar = 50 μm. (C, F, I, L, O) Expression of IL-1b at day 21 after SE. (B) Expression of IL-1b at the hippocampus at day 21 after SE in low-power magnification. Scale bar = 1 mm. (E) CA1 region at day 21 after SE in intermediate power magnification. (H) CA3 region at day 21 after SE in intermediate power magnification. F, I; scale bar = 100 μm. (F) CA1 region at day 21 in high power magnification. (N) CA3 region at day 21 after SE in high power magnification. Reactive astrocyte-like cells emerging in CA1 showed IL-1b immunoreactivity on day 7. This immunointensity increased in proportion to progressive hypertrophy until day 21. L, O; scale bar = 50 μm.

Article Snippet: For the immunohistochemical study, a commercially available rabbit polyclonal anti-IL-1b antibody (diluted 1:500; sc-1251, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used.

Techniques: Control, Expressing

Journal: Japanese Clinical Medicine

Article Title: Dynamic Change in Cells Expressing IL-1β in Rat Hippocampus after Status Epilepticus

doi: 10.4137/jcm.s13738

Figure Lengend Snippet: Figure 3. Double-label immunofluorescence staining of IL-1b and GFAP. Co-localization of IL-1b (red, A) and GFAP (green, B) is shown with an immunofluorescence method. Co-localization is visualized in yellow in the merged image (C). Double-label fluorescent immunohistochemistry clarified that reactive astrocytes expressed IL-1b. Scale bar = 50 μm.

Article Snippet: For the immunohistochemical study, a commercially available rabbit polyclonal anti-IL-1b antibody (diluted 1:500; sc-1251, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used.

Techniques: Immunofluorescence, Staining, Immunohistochemistry

Journal: Japanese Clinical Medicine

Article Title: Dynamic Change in Cells Expressing IL-1β in Rat Hippocampus after Status Epilepticus

doi: 10.4137/jcm.s13738

Figure Lengend Snippet: Figure 4. Total IL-1b expression in the hippocampus after SE. The total expression level IL-1b in the hippocampus measured by using Luminex technology was significantly elevated from day 1 after SE and maintained till day 21 (P 0.01). Asterisks (*) and daggers (†) indicate significant differences (P 0.05 and P 0.005, respectively) from the value for the control group.

Article Snippet: For the immunohistochemical study, a commercially available rabbit polyclonal anti-IL-1b antibody (diluted 1:500; sc-1251, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used.

Techniques: Expressing, Luminex, Control

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Neurite Outgrowth by Interactions between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-associated Protein Superior Cervical Ganglia Clone 10

doi: 10.1074/jbc.M109.064113

Figure Lengend Snippet: Interaction between JLP and stathmin family members. A, cell lysates derived from COS-7 cells overexpressing JLP-S and HA-tagged stathmin, SCG10, and SCLIP were subjected to pulldown assays using S-protein-agarose beads, and the precipitates were analyzed by SDS-PAGE, followed by Western blotting with an anti-HA antibody (middle panel). The expression of HA-tagged stathmin, SCG10, and SCLIP, as well as the precipitated S-tagged JLP (JLP-S), are shown in the top and bottom panels. B and C, schematic representation of the deletion and domain mutants of JLP, respectively. D and E, domain mapping of the JLP-SCG10 interaction. A series of deletion mutants (B) and domain mutants (C) of JLP were co-transfected into COS-7 cells with HA-SCG10 (top panel) and subjected to precipitation using S-protein-agarose. Precipitates were analyzed by SDS-PAGE, followed by Western blotting with an anti-HA antibody. The expression of HA-SCG10 and the precipitated JLP mutants are shown in the top and bottom panels.

Article Snippet: Generation of a Rabbit Polyclonal Anti-SCG10 Antibody Because the N-terminal region of SCG10 is insoluble, a truncated form that is devoid of the first 35 amino acids was used as an antigen to raise a rabbit polyclonal anti-SCG10 antibody (Rockland Immunochemicals).

Techniques: Derivative Assay, SDS Page, Western Blot, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Neurite Outgrowth by Interactions between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-associated Protein Superior Cervical Ganglia Clone 10

doi: 10.1074/jbc.M109.064113

Figure Lengend Snippet: SCG10 binds to JLP only in its full-length form. A, schematic representation of the domain mutants of SCG10. B, mapping JLP-interacting domain of SCG10. A series of domain mutants of SCG10 (top panel) were co-transfected into COS-7 cells with JLP-S and subjected to precipitation using S-protein-agarose. Precipitates were analyzed by SDS-PAGE, followed by Western blotting with an anti-HA antibody. The expression of HA-SCG10 mutants and the precipitated JLP are shown in the top and bottom panels. C, the interaction between JLP and SCG10 during NGF-induced differentiation of PC12 cells. An immunoprecipitation assay using an anti-SCG10 or JLP antibody was performed using cell lysates derived from the PC12 cells treated with NGF for 3 days. The immunoprecipitates together with the lysate were analyzed using the antibodies against JLP or SCG10. D, a time-course study of the interaction between JLP and SCG10. Immunoprecipitation assays were performed as in C using cell lysates derived from PC12 cells treated with NGF at various time points as indicated. The lysates were immunoprecipitated with control antibody (cont. Ab) or anti-SCG10 antibody (SCG10 Ab). Western blot analysis was performed on the lysates and the immunoprecipitates using specific antibodies against JLP and SCG10.

Article Snippet: Generation of a Rabbit Polyclonal Anti-SCG10 Antibody Because the N-terminal region of SCG10 is insoluble, a truncated form that is devoid of the first 35 amino acids was used as an antigen to raise a rabbit polyclonal anti-SCG10 antibody (Rockland Immunochemicals).

Techniques: Transfection, SDS Page, Western Blot, Expressing, Immunoprecipitation, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Neurite Outgrowth by Interactions between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-associated Protein Superior Cervical Ganglia Clone 10

doi: 10.1074/jbc.M109.064113

Figure Lengend Snippet: The role of JLP in regulating SCG10 phosphorylation in PC12 cells. A, the phosphorylation level of endogenous SCG10 in PC12 cells in response to NGF treatment. PC12 cells were transfected with control siRNA (C) or JLP siRNA (J), followed by NGF treatment. Two days after the treatment, the cultures were incubated with [32P]orthophosphate for 2 h. Lysates from the radiolabeled cells were subjected to immunoprecipitation analysis using an anti-SCG10 antibody. The immunoprecipitates were resolved by SDS-PAGE and subjected to autoradiography. B, quantitation of SCG10 phosphorylation in the presence or absence of JLP. The top and bottom SCG10 protein bands indicated in A are quantitated separately. The basal phosphorylation levels of SCG10 in intact PC12 cells were defined as 1 unit.

Article Snippet: Generation of a Rabbit Polyclonal Anti-SCG10 Antibody Because the N-terminal region of SCG10 is insoluble, a truncated form that is devoid of the first 35 amino acids was used as an antigen to raise a rabbit polyclonal anti-SCG10 antibody (Rockland Immunochemicals).

Techniques: Transfection, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Neurite Outgrowth by Interactions between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-associated Protein Superior Cervical Ganglia Clone 10

doi: 10.1074/jbc.M109.064113

Figure Lengend Snippet: JNK activity is required for the NGF-induced neurite outgrowth of PC12 cells. A, the effect of JNK inhibitor SP600125 on NGF-induced differentiation of PC12 cells. PC12 cells were incubated with vehicle (DMSO) or the JNK inhibitor (10 μm) 2 h before NGF treatment. Three days after the treatment, randomly selected fields were photographed in the indicated culture plates. B, changes in the phosphorylation level of SCG10 as a result of SP600125 treatment. PC12 cells were incubated with vehicle (DMSO) or the JNK inhibitor (10 μm) 2 h before NGF treatment. Three days after the treatment, the cultures were incubated with [32P]orthophosphate for 2 h. Lysates from the radiolabeled cells were immunoprecipitated using an anti-SCG10 antibody. The immunoprecipitates were resolved by SDS-PAGE and subjected to autoradiography. Phosphorylated levels of the top and bottom bands of SCG10 were quantitated. The phosphorylated levels of SCG10 of the control samples were set at 100. C, protein analysis of the lysates derived from the cells treated with vehicle (DMSO) or SP600125. PC12 cells were incubated with vehicle (DMSO) or the JNK inhibitor (10 μm) 2 h before NGF treatment. Three days after the treatment, the lysates were analyzed by Western blotting using specific antibodies against phospho-JNK, JNK, SCG10, JLP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Article Snippet: Generation of a Rabbit Polyclonal Anti-SCG10 Antibody Because the N-terminal region of SCG10 is insoluble, a truncated form that is devoid of the first 35 amino acids was used as an antigen to raise a rabbit polyclonal anti-SCG10 antibody (Rockland Immunochemicals).

Techniques: Activity Assay, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Derivative Assay, Western Blot