Fig 2: GSK3a/ß-mediated phosphorylation leads to Arc degradation in neurons. (A) Quadruple (4A) mutant of Arc is more stable than wildtype protein in NMDA-stimulated neurons. The figure shows the Western blot analysis of HA-tagged Arc overexpressed in cortical neurons. Cortical neurons were transduced with lentiviral vectors that carried wildtype SARE-Arc or SARE-Arc4A. Twenty-one hours posttransduction, the cells were treated with NMDA for 4 h, and anisomycin was added for the times indicated. (B) Quantification of Western blot results from (A). Next, relative decrease in HA-Arc expression was calculated as a ratio of HA-Arc level at a given time point (45 or 90 min of anisomycin treatment) to HA-Arc level at the “time 0”, when anisomycin was added to neurons pretreated with NMDA. Decrease in Arc and Arc 4A levels was compared separately for 45 or 90 min of anisomycin treatment and the difference was evaluated with t-test. The data are expressed as mean ± SEM (n = 5 independent cultures). *p < 0.05, **p < 0.01 (t-test). (C) Unphosphorylatable mutant of Arc is insensitive to GSK3ß- mediated degradation. The figure shows representative confocal images of HA-tagged Arc expression in hippocampal neurons that were co-transfected with ß-actin 16pl vector or GSK3ß S9A and wildtype SARE-Arc or the 4A mutant together with SARE-mCherry plasmid for 20 h and stimulated with NMDA for 4 h. Images were transformed to “blue to yellow” pseudo color mode to visualize differences in immunofluorescence intensity. Scale bar = 10 µm. (D) Quantitative analysis of Arc expression. HA immunofluorescence was measured and normalized to mCherry fluorescence. For each kind of transfection, the normalized expression of HA-tagged Arc in NMDA-treated neurons was compared with Arc expression in control cells. Sixty to seventy cells per condition from three independent cultures were analyzed. **p < 0.01, ***p < 0.001; ns, not significant (Mann-Whitney test).

Fig 3: ALFA-ArcNbs for immunoblotting and immunoprecipitation analysis of endogenous Arc. A Rat cortical tissues were lysed in RIPA buffer. Different amounts of lysates were subject to SDS-PAGE followed by immunoblotting using ALFA-ArcNbs (B5, B12, C11, D4, E5, and H11) and conventional anti-Arc antibodies as reference. Immunoreactive bands corresponding to Arc were detected around 50 kDa (lane 1, 30 µg; lane 2, 60 µg). B CCh (100 µM, 1 h)-treated SH-SY5Y cells were lysed and subjected to immunoprecipitation using ALFA-ArcNb H11 and ALFA Selector™ followed by SDS-PAGE and immunoblotting using anti-Arc antibody. NC, negative control (ALFA SelectorST resin without ALFA-ArcNb H11. C HFS was applied unilaterally to the perforant path input to the dentate gyrus (DG) of anesthetized rats to induce LTP. At 2 h post-HFS, DG tissue was collected from the ipsilateral (Ipsi) and contralateral (Contra) DG, and immunoprecipitation analysis was performed as described in panel B. Strong enrichment of Arc was obtained upon addition of ALFA-ArcNb H11 (open arrows). The signal in negative control is attributed to non-specific Arc binding to agarose resin

Fig 4: H2A.Z.2 facilitates priming of the Arc promoter. A, Representative Western blotting of whole-cell lysate showing Ac-H2A.Z levels in neurons depleted of H2A.Z hypervariants as indicated. N = 3. B, Graphical map (not to scale) depicting relative position of ChIP primers near Arc TSS. C, Quantification of Ac-H2A.Z (normalized by total H2A.Z) binding to Arc TSS region in neurons treated as indicated, determined by ChIP. N = 4. Two sets of data in statistical comparison are represented by the position and the color of asterisk(s). D, ChIP data demonstrating binding of RPB1 to the Arc TSS region in control neurons or H2A.Z hypervariant-depleted neurons as indicated. N = 4. Note the enrichment of RPB1 at the TSS (primer 2) in control neurons (black) indicating promoter-proximal RNA Pol II pausing. E, ChIP data demonstrating binding of pSer5-RPB1 to the Arc TSS in control and hypervariant-depleted neurons. N = 3. *p < 0.05 and **p < 0.01.

Fig 5: AP-2 is required for Arc-dependent homeostatic scaling. A, Average mEPSC waveforms from an untransfected neuron cultured in control conditions, from an untransfected neuron exposed to bicuculline and from a µ2-shRNA2 expressing neuron that has been incubated in bicuculline (40 µm; 48 h). Note that the bicuculline-induced down regulation of the mEPSC amplitude was reduced in AP-2 depleted cells (µ2-shRNA2 expressing cells).The untransfected neuron and the neuron expressing µ2-shRNA2 that were cultured in the presence of bicuculline were neighbors in the same dish, while the untransfected neuron cultured in control conditions was from the same preparation. B, Cumulative amplitude distribution for untransfected neurons cultured in control conditions (black line, n=10 neurons), untransfected neurons incubated in bicuculline (red line, n=15 neurons), µ2-shRNA2 expressing cells incubated in bicuculline (blue line; n=6 neurons) and cells transfected with n.c. shRNA incubated in bicuculline (green line; n=5 neurons). C, Bar chart plotting the mean mEPSC amplitude for the cells shown in B. Incubation in bicuculline significantly reduced the mean mEPSC amplitude (from 17.3 ± 1 pA to 11.9 ± 0.2 pA; p=0.0001). Expression of shRNA2 significantly increased mEPSC amplitude in bicuculline (14.38 ± 0.16 pA; p=0.0001), whereas n.c shRNA had significantly less effect (12.9 ± 0.28 pA; p=0.007). D, Bar chart plotting the mean interval between mEPSCs for the cells in B and C. The error bars in C and D are SEM. ***p<0.001, **p<0.01. Statistical significance was tested using the Mann–Whitney test.