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. 2010 Nov 4;6(11):e1001200. doi: 10.1371/journal.pgen.1001200

Figure 5. Levels of UNC-104 motor in cargo binding variant alleles depends on ubiquitination.

Figure 5

Ubiquitination is reduced using the mutant uba-1(it129ts) at 22°C. (A1,A2) Anti-UNC-104 western blots. (A1) Total UNC-104 levels increase in unc-104(e1265) upon reducing ubiquitination. The same western blot with two different exposures has been provided for unc-104(e1265). (A2) UNC-104 levels increase in unc-104(e1265tb107) and in unc-104(e1265tb120) upon reducing ubiquitination. (B) Anti-UNC-104 immunoreactivity in all unc-104 cargo-binding variants in uba-1(it129ts) background. Upon reducing ubiquitination, UNC-104 levels increase in unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) in synapse rich regions of the nerve ring (arrow) and ventral cord (arrowhead). (C) Localization of SNB-1::GFP cargo markers in neuromuscular junctions observed using juIs1 in unc-104 cargo binding variant alleles. Upon reducing ubiquitination, cargo marked by synaptobrevin increases at synapses in unc-104(e1265tb107) and in unc-104(e1265tb120). Arrowhead points to ventral cord motor neuron synaptic puncta. Arrow points to the cell body. (D) Mean fluorescence intensity of neuromuscular junction synaptic puncta marked by synaptobrevin::GFP. The intensity of synaptic puncta in unc-104(e1265tb107) and unc-104(e1265tb120) increases upon blocking ubiquitination. (n = 10 animals and *p<0.05). Data represented as mean ± SEM. (E) Aldicarb resistance assay in the UNC-104 PH domain variant alleles. Time for aldicarb induced paralysis decreases in unc-104(e1265tb107) and in unc-104(e1265tb120) upon block in ubiquitination. n = 30 animals, done three times independently. Data represented as mean ± SEM (*p = 0.005). The alleles unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) are labeled in the figure by the respective protein changes they encode, namely D1497N, D1497N R1501Q and D1497N M1540I.