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. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: Neurobiol Dis. 2008 Jul 3;32(1):26–36. doi: 10.1016/j.nbd.2008.06.013

Figure 6. Expression and distribution of TRIP8b was unchanged in TLE.

Figure 6

A–B, Rabbit α-TRIP8b antibody is specific in biochemical and immunohistochemical assays. A, Protein extracts from Cos-7 cells transfected with a TRIP8b-GFP-expressing plasmid or rat brain were separated by SDS-PAGE and blotted with α-TRIP8b antibody. Our custom antibody detected a ~110 kD band in transfected Cos-7 cells and band of ~78 kD in rat brain, consistent with the predicted size of the GFP-fusion and native protein, respectively. B, Parasagittal sections of rat brain were immunolabeled with α-TRIP8b antibody. Note the strong immunoreactivity in SLM of hippocampal area CA1 (arrow) and layer I-II of cortex (arrowheads). Scale bars: 200µm. (SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum; SLM: stratum lacunosum moleculare). C, TRIP8b interacts with h channel subunits in the rat brain. Membrane fractions of rat brain extracts were generated and coimmunoprecipitation performed using antibodies against h channel subunits (Top, gp α-HCN1 antibody, gp α-HCN2 antibody, gp α-HCN4 antibody) or α-TRIP8b antibody (bottom). Immunoprecipitation using preimmune serum (PI) served as a negative control. D, Protein expression levels of TRIP8b were not altered in the CA1 hippocampus of SE animals. (Top) Hippocampal area CA1 was sub-dissected and membrane extract was generated. Proteins were separated by SDS-PAGE and immunoblotted using α-TRIP8b antibody or ms α-α-tubulin antibody. Protein expression levels of TRIP8b were quantitated by densitometry and normalized with the level of α-tubulin (as a loading control). (Bottom) Graphing the relative density of TRIP8b of SE and non-SE animals shows that TRIP8b protein expression levels were not significantly changed at either 1 d (1 d SE; 104.4±10.8% of 1 d control, n=4; p>0.7) or 28 d (28 d SE; 95.7±3.7% of 28 d control, n=4; p>0.7) after SE. E, Distribution of TRIP8b in CA1 hippocampus was unaltered after SE. 50µm parasagittal sections of control (left) and 28d SE (right) brains were immunolabeled with α-TRIP8b antibody and visualized with DAB staining. TRIP8b distribution was not changed in 28 d as compared to control CA1.