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. 2019 Oct 10;17(10):e3000461. doi: 10.1371/journal.pbio.3000461

Fig 3. DIP2A interacts with cortactin and modulates cortactin acetylation (ac-cortactin).

Fig 3

(A) Interaction between endogenous DIP2A and cortactin from murine brain lysates. (B and C) Interaction between endogenous DIP2A, cortactin, and SHANK3 from murine brain lysates. (D) Representative image showing colocalization of DIP2A (green) and cortactin or SHANK3 (red) in cultured neurons. (E) Western blot showing DIP2A and cortactin, and SHANK3 in the cortical PSD composition (lane 4). Total, total homogenate; P2, crude synaptosomes; SV, crude synaptic vesicle fraction. (F) Western blot and bar graph showing Dip2a deletion reduced ac-cortactin without altering levels of total cortactin and phosphorylated cortactin (p-cortactin) (P56, males; n = 4 mice per genotype; data from 3 independent experiments). Levels of ac-cortactin and tubulin (ac-tub) were normalized to total cortactin and α-tubulin, respectively. The ratio in WT mice was set to 100% (ac-cortactin, t10 = 2.320, *P = 0.0428; p-cortactin, t10 = 0.8637, n.s. P = 0.4080; cortactin, t10 = 1.712, n.s. P = 0.1177; ac-tub, t10 = 0.1943, n.s. P = 0.8498). (G) Dip2a deletion reduced ac-cortactin without affecting total or p-cortactin levels in cultured cortical neurons (DIV15) (data from 3 independent experiments, approximately 5 × 106 cells per experiment; ac-cortactin, t10 = 3.258, **P = 0.0086; p-cortactin, t10 = 0.1551, n.s. P = 0.8798; cortactin, t10 = 0.4124, n.s. P = 0.6888; ac-tub, t10 = 0.1507, n.s. P = 0.8832). (H) Western blot showing overexpressed DIP2A in cultured cells with elevated ac-cortactin level. (I and J) Normalized grayscale value in (H) (data from 3 independent experiments and presented as mean ± SEM; one-way ANOVA; I, F3,20 = 19.13, post hoc LSD, *P = 0.0199, **P = 0.0020 and 0.0016, respectively; J, F3,20 = 17.76, post hoc LSD, n.s. P = 0.3227, ***P < 0.0001, *P = 0.0379). The underlying data for this figure can be found in S1 Data. ac-cortactin, cortactin acetylation; DIP2A, disconnected-interacting protein homolog 2 A; DIV, day in vitro; FLAG, FLAG tag with the sequence DYKDDDDK; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, HA-tag (YPYDVPDYA); HDAC6, histone deacetylase 6; IgG, immunoglobulin G; IP, immunoprecipitation; KO, knockout; LSD, least significant difference; n.s., no significance; p-cortactin, phosphorylated cortactin; PSD, postsynaptic density; P2, crude synaptosomes; SHANK3, SH3 and multiple ankyrin repeat domains 3; SV, crude synaptic vesicle fraction; TSA, trichostatin A; tub, tubulin; WT, wild-type.