(A) Pulse chase of SAX-3(WT) and SAX-3(P37S) in the presence of EBAX-1 WT or ΔBox. HEK293T cells expressing indicated constructs were treated with 1 μM cycloheximide (CHX) for 0, 2, 4, or 6 hours (h) before collection. An equal amount of protein from each sample was loaded to SDS-PAGE and immunoblotted with mouse anti-Flag, V5 and α-tubulin antibodies.
(B) Representative images of Dendra-tagged SAX-3(WT) and SAX-3(P37S) in AVM neurons at 0 and 7 h after photoconversion. SAX-3(P37S) showed a faster degradation rate (b3-b4) than SAX-3(WT) (b1-b2). The fluorescence intensity is indicated as a pseudocolor scale. Scale, 2 μm.
(C) Comparison of SAX-3(WT)::Dendra and SAX-3(P37S)::Dendra fluorescence intensity 7 h after photoconversion at 20°C and 22.5°C. The yellow area indicates the range of SAX-3(WT) samples and the blue area covers mutant samples with faster degradation rates than WT samples.
(D) The effects of ebax-1(ju699) on the degradation of SAX-3(WT)::Dendra and SAX-3(P37S)::Dendra in AVM neurons. Data represent means ± SEM. *p<0.05, **p<0.01 and ***p<0.001 relative to indicated controls; t-test.