This is consistent with previous that demonstrated that JIP3 can mediate retrograde transport of JNK in response to axonal injury through interactions Cabozantinib molecular weight with the P150 glued subunit of the dynein motor protein complex, and it is likely that DLK JNK interaction with JIP3 mediates retrograde transport of JNK after NGF withdrawal also. It is also probable that the signaling specificity downstream of DLK is mediated by activation of only a subset of the three JNK genes in mouse, all of which are expressed in embryonic neurons. The phenotypes observed in JNK null rats argue that JNK2 and JNK3 are largely responsible for your JNKmediated neuronal deterioration, at the very least in the context of damage. More over, JIP3 has been proven to preferentially connect to JNK3 over other JNK isoforms Figure 8. A model for the regulation of developmental apoptosis and axon degeneration by DLK. A complex containing DLK, JIP3, JNK, and possibly additional proteins is activated inside the distal axon after resonance NGF withdrawal and in caspase dependent neuronal apoptosis and axonal degeneration. . Apoptosis happens via phosphorylation of c Jun and retrograde transport of JNK, but axon deterioration doesn’t require c Jun and is mediated by different JNK targets. 762 JCB VOLUME 194 # 5 2011 using recombinant protein consisting of DLK A597 P888, provided by S. Hirai or Genentech, Hirai et al., 2005. Slides were mounted in Fluoromount H, incubated for 1 h at room temperature with Alexa Fluor conjugated secondary antibodies accompanied by 3 PBS clears, and washed three times in PBS. Staining of tissue was performed using the protocol above but with PBS containing 5% normal goat serum and 0.. 1000 Triton X 100 on 20 um transverse sections cut on a cryostat.. The antibodies applied were pan Trk, activated caspase 3, HB9, and Alexa JZL184 clinical trial Fluor conjugated secondary antibodies. . For wholemount embryo neurofilament discoloration, embryos were eviscerated, fixed in 4% PFA, and stained with rabbit anti Neurofilament antibody using the same protocol as described above, except that most antibody incubations were overnight, and buffers included 0. Four weeks Triton X 100. Western blotting and IP DRG cultures were lysed in 100 ul Triton X 100 lysis buffer for 30 min at 4 C. Due to the limited amount of protein prepared from DRGs, protein was precipitated applying TCA and then washed with acetone 3 times to get rid of the TCA. The pellet was dried and re-suspended in 1 SDS NuPAGE running buffer containing a reducing agent. The amount of protein in samples was quantified by Western blotting for tubulin. Similar amounts of protein were then loaded on 4 127-inch Bis Tris ties in and subjected to standard immunoblotting procedures. Major antibodies used for Western blotting were just like those used for immunocytochemistry. Mark photographs were taken and quantified using the VersaDoc system.