Figure 4. Triad3A Regulates Arc Stability and Turnover.
(A) Enhanced Arc induction upon depletion of Triad3A in hippocampal neurons. Hippocampal neurons were transduced with control or Triad3 lentiviral shRNAs and treated with either TTX (2 μM) or 4AP/Bic. Cell lysates were resolved by SDS-PAGE and immunoblotted with Arc or Triad3A specific antibodies. Note that Triad3A knockdown results in higher Arc levels after 4AP/Bic induction.
(B) Means ± SEM of Arc levels under the indicated conditions displayed as a fold change relative to the Scr control. *p < 0.05, n = 3.
(C) Loss of Triad3A increases Arc half-life in neurons. Hippocampal neurons were incubated with control lentiviral shRNA or Triad3-shRNA and treated with either TTX (2 μM) or 4AP/Bic. Neurons were then incubated in 35S-Cys/Met for metabolic labeling and pulse-chase analysis, upper panel. Immunoblots of radiolabeled extracts using anti-Arc, anti-Triad3A, and anti-tubulin antibodies are shown in the lower blots. Note the reduced turnover of Arc (35S-Arc) as well as the increased remaining total Arc in neurons lacking Triad3A.
(D) Quantification of Arc half-life 3 h after 4-AP/Bic treatment displayed as a fold change relative to Scr control. Data represent means ± SEM. *p < 0.05, n = 3.
(E) Immunostaining for Arc upon Triad3A overexpression or downregulation. DIV14 hippocampal neurons were transfected with cDNA encoding GFP along with either myc-Triad3A or Triad3-shRNA for 72 h. Cells were treated with TTX or 4-AP/Bic for 4 h prior to fixation and immunostaining for Arc. Arrows indicate reduced Arc levels in a Triad3A overexpressing neuron. Arrowheads indicate elevated Arc in neurons expressing Triad3 shRNA. Scale bars, 25 μm.
(F) Data represent means ± SEM of Arc staining in neurons expressing GFP-Triad3A, Triad3-shRNA, or shRNA-resistant Triad3A (Triad3A*) from (E). AFU, arbitrary fluorescence units. *p < 0.01, n = 6–22.
(G) Triad3A is required for activity-induced Arc ubiquitination in neurons. Cortical neurons transduced with Scr or Triad3 (T3) shRNA lentivirus and incubated with TTX or 4AP/Bic. Arc was immunoprecipitated from cell extracts under denaturing conditions. Immunoprecipitates were resolved by SDS-PAGE and probed with an anti-K48-linkage specific ubiquitin antibody or anti-Arc antibody. 4AP/Bic induced Arc polyubiquitination (lane 3) that was prevented by Triad3-shRNA (lane 4).