Fig 2: Nlk destabilizes nuclear Tfeb via proteasomal degradation.(A and B) Western blots of N2a whole-cell protein lysates following transfection with Flag-Nlk WT or Flag-Nlk KN (A). Tfeb levels decreased upon Nlk overexpression in a kinase activity–dependent manner, quantified in B (n = 3 biological replicates). (C and D) Western blots of WT N2a cells transfected with Flag-Nlk WT or Flag-Nlk KN showed a kinase activity–dependent decrease of nuclear Tfeb levels (C), quantified in D (n = 3 biological replicates). (E and F) Western blots of N2a whole-cell protein lysates following transfection with Flag-Nlk WT or Flag-Nlk KN, with DMSO vehicle or MG132 treatment (E). Tfeb levels decreased upon Nlk overexpression in a kinase activity–dependent manner, but this destabilization was not observed in the presence of the proteasome inhibitor MG132, quantified in F (n = 3 biological replicates). (G–J) Western blots of subcellular fractionated N2a protein lysates following transfection with Flag-Nlk WT or Flag-Nlk KN, with DMSO or MG132 treatment (G). Only nuclear Tfeb levels were reduced upon Nlk overexpression in a kinase activity–dependent manner, and this decrease was dependent on the proteasomal degradation pathway, quantified in H. Phosphorylation of Tfeb at S122 and S142 was not affected by Nlk overexpression, quantified in I and J (n = 4 biological replicates). One-way ANOVA analyses (B, D, F, and H–J) were performed, and data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.