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. 2005 Jun 29;25(26):6092–6104. doi: 10.1523/JNEUROSCI.0707-05.2005

Figure 6.

Figure 6.

Interaction of caspase-8 with dynactin p150Glued and the dynein intermediate chain after NMDA lesion. A, NMDA was administered unilaterally into the olfactory bulb, and dissection of the olfactory neuraxis into four regions spanning the caudal end of the olfactory bulb to the rostral end of the OE was performed. B, Dynactin p150Glued was immunoprecipitated from OB and OE protein extracts containing pooled, microdissected regions of the NMDA-lesioned and unlesioned olfactory neuraxis. The interaction with caspase-8 (α-casp8) was analyzed by Western blots using rabbit polyclonal antibodies recognizing the p17 subunit (MF438). Dynactin p150Glued (α-p150Glued) coimmunoprecipitated with full-length caspase-8 (on both lesioned and unlesioned sides), but it was seen only on the lesion side to complex with the p17 large catalytic subunit of cleaved caspase-8 (arrow). prG, Protein-G beads only. C, Dynactin p150Glued and dynein intermediate chain (IC) were readily detected in total cell lysates of the olfactory epithelium (TOE) or olfactory bulb (TOB). When caspase-8 (α-casp8) was immunoprecipitated from these lysates, it was found to be complexed with both dynactin p150Glued and dynein IC (top two panels, OE and OB lanes). Similarly, immunoprecipitates of dynein IC (bottom panel, OE and OB lanes) coimmunoprecipitated dynactin p150Glued. prG, Protein-G beads only. D, Dynactin p150Glued immunoprecipitates were probed by Western blot analysis for the p17 subunit, and active caspase-8 associated with dynactin p150Glued was quantified in the four sample areas specified in A by densitometric scanning (n = 4-5). The complex of active caspase-8 with dynactin p150Glued increased dramatically (compared with contralateral, unlesioned side) at 24 h after NMDA treatment, and this complex shifted from the bulb to the periphery between 24 and 48 h after NMDA treatment. co-IPed, Coimmunoprecipitated. Concomitantly, at 24 h (E), active caspase-8 immunolabeling could be detected in ORN axons in both the NFL of the bulb and the OE turbinates in the periphery. E, External plexiform; Gl, glomerular.