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. 2011 May 18;31(20):7424–7440. doi: 10.1523/JNEUROSCI.0936-11.2011

Figure 5.

Figure 5.

TRIP8b deletion reduces HCN1 and HCN2 protein levels without affecting HCN1/HCN2 heteromerization, upregulation of the unfolded protein response protein BiP (Grp78), or sequestration of HCN1 protein in Golgi apparatus. A, qPCR from hippocampal mRNA shows absence of Pex5l mRNA in TRIP8b−/− mice but no significant differences in mRNA coding for HCN channel subunits (***p < 0.001, n = 3, unpaired t test). B, Immunoblots of lysates from subdissected hippocampal area CA1 show decreased HCN1 and HCN2 subunit protein in TRIP8b−/− mice (*p < 0.05, ***p < 0.001, n = 4–6, unpaired t test). C, Immunoprecipitation (IP) of HCN1 followed by immunoblotting (IB) for HCN2 (left, top panels) and IP of HCN2 followed by IB for HCN1 (left, bottom panels) from hippocampal lysates was not significantly different in TRIP8b−/− mice compared with TRIP8b+/+ mice (n = 3 mice/genotype; p > 0.10, unpaired t test). Input is 5% of total lysate immunoprecipitated. The bottom blot shows shorter exposure of top blot, used to quantify the IP fraction compared with the input quantified from top blot. NS, Not significant. D, Immunoblotting of hippocampal lysates from TRIP8b+/+ and TRIP8b−/− mice with antibodies against BiP (Grp78), TRIP8b, and α-tubulin. E, Immunohistochemistry for HCN1 (green) and GM130 (marker of Golgi apparatus; red) in CA1 stratum pyramidale from TRIP8b+/+ and TRIP8b−/− mice demonstrates no change in colocalization, suggesting TRIP8b is not required for HCN1 transit from Golgi apparatus. Scale bar, 10 μm.