Fig. 5: Id2 competitively interrupts the association of Sirt2 with α-tubulin.

A, B GST–Id2 and mammalian GFP–Sirt2 fragments (A) transfected cells were subjected to GST pull-down assay and immunoblotting as indicated. C, D Flag–Sirt2 and mammalian GST–α-tubulin fragments (C) transfected cells were subjected to GST pull-down and immunoblotting as indicated. E, F HEK293T cells were transfected with 0, 1, 3 μg of GFP–Id2 (E) or Flag–Sirt2 (F) with GST–α-tubulin, and lysates were subjected to GST pull-down and immunoblotting. The bar graphs represent the quantification of Sirt2 or Id2–α-tubulin binding. G, H Primary hippocampal neurons were transfected as indicated and the cell lysates were subjected to GST pull-down assay. I Proximity ligation assay (PLA) was conducted with scr, si-Id2, or si-Sirt2 expressing cells. Confocal images were shown tubulin-Id2 PLA staining (left) and tubulin-Sirt2 PLA staining (right). Nuclei were stained by DAPI (blue). Scale bar 5 μm. Quantification of tubulin-Id2 or tubulin-Sirt2 PLA puncta is shown as a bar graph. **p < 0.005, ***p < 0.0005, ****p < 0.0001. All values are mean ± SEM and the representative images shown are from at least three independent experiments.