Skip to main content
. 2022 Apr 26;11:e74277. doi: 10.7554/eLife.74277

Figure 2. Neuronal activity bidirectionally modulates the phosphorylation state of Shank3.

(A) The experiment protocol for extraction of Shank3 from rat cultured neocortical neurons for further quantitative mass spectrometry (MS) or Western blot analyses. (B) Volcano plot of quantitative MS data showing Shank3 residues that were differentially phosphorylated in tetrodotoxin (TTX)-treated samples compared to untreated controls. The log2 values of fold changes, if below zero, indicated hypophosphorylation (paired t-test: S1586, adjusted p=0.034142, S1614/5, 0.014444). (C) Top: diagram showing the location of S1586 and S1615 within the rat Shank3 protein. Functional domains: ANK = ankyrin repeat; SH3 = SRC homology 3; PDZ = PSD-95/Disc Large/ZO-1; Pro-rich = proline rich; SAM = sterile alpha motif. Bottom: homology comparison of sequences flanking rat S1586 and S1615 (matching mouse S1539) across species (human Shank3: NP_001358973.1; rat Shank3: NP_067708.2; mouse Shank3: UniprotKB: Q4ACU6.3). Phosphosites of interest are labeled in red; the only residue not conserved is shown in blue. (D, E) Representative Western blot using an antibody specific for phosphorylated S1615, showing changes in Shank3 phosphorylation after 10 min (D) or 24 hr (E) treatment with TTX or picrotoxin (PTX). (F) Quantification of the fold change of Shank3 S1615 phosphorylation in (D). Dashed line indicates the baseline untreated control (one-sample t-test: TTX, ***p=0.0005, PTX, **p=0.0035, n = 5 and 10 biological replicates, respectively). (G) Quantification of the fold change of Shank3 S1615 phosphorylation in (E) (one-sample t-test: TTX, ****p<0.0001, PTX, p = 0.6336, n = 7 and 7 biological replicates, respectively). Solid colored horizontal lines indicate the mean, and error bars represent SEM. Also see Figure 2—figure supplement 1, Figure 2—source data 1, and Figure 2—source data 2.

Figure 2—source data 1. Source data for Figure 2.
Figure 2—source data 2. Source blot images for Figure 2.

Figure 2.

Figure 2—figure supplement 1. Validation of the pS1615 antibody.

Figure 2—figure supplement 1.

(A) Western blot image showing the intensity of pS1615 in HA-tagged wild-type Shank3 or the S1586A/S1615A (AA) mutant. One set of replicates were treated with the phosphatase inhibitor, okadaic acid (OKA, 500 nM) for 30 min before lysis. Untr., untransfected control; n = 3 biological replicates. (B) Western blot image showing the intensity of pS1615 in HA-tagged wild-type Shank3 with and without the treatment of lambda phosphatase (n = 4 biological replicates). Also see Figure 2—figure supplement 1—source data 1.
Figure 2—figure supplement 1—source data 1. Source blot images for Figure 2—figure supplement 1.