mouse anti glua1  (Novus Biologicals)

Journal: Nature Communications

Article Title: Elevated synaptic PKA activity and abnormal striatal dopamine signaling in Akap11 mutant mice, a genetic model of schizophrenia and bipolar disorder

doi: 10.1038/s41467-025-66504-2

Figure Lengend Snippet: a Schematic of AKAP11 immunoprecipitation (IP) and its most notable, known interactors. b Immunoblot of AKAP11 in IPs of anti-AKAP11 and control IgG in 12-week-old (12wk) WT or Akap11 -/- cortical tissue. This experiment was repeated in >5 independent experiments, with similar results. c Volcano plot comparing Log Fold-Change (Log FC) against P value for AKAP11 IP in WT vs Akap11 -/- , displaying all proteins detected by coIP-MS. A moderated two-sample t-test was applied to the sample group datasets. Proteins that were enriched (red) by anti-AKAP11 IP from WT versus Akap11 -/- cortical extracts. Enriched proteins, defined by having a nominal P value < 0.05 and Log FC > 0, are colored red. Downregulated proteins are mostly antibody-related artifacts and can be found in Supplementary Data . d Venn diagram comparing previously published AKAP11 interactions with the brain interactome in this study. Full results are displayed in Supplementary Data . e Immunoblot of AKAP11, PKA subunits and control proteins, in IPs of AKAP11, GluA1 or IgG control in WT or Akap11 -/- cortical lysate. This experiment was repeated in >3 independent experiments, with similar results. f Immunoblot of AKAP11 in IPs of PKA subunits, in WT cortical lysates. This experiment was repeated in >2 independent experiments, with similar results. g–j Immunoblot with indicated antibodies, in IPs of anti-GSK3α, GSK3β, VAPA, VAPB, AKAP11, P62 or DYRK1a in WT or Akap11 -/- cortical lysate. These experiments were repeated in at least 2 independent experiments, with similar results. k Gene set enrichment analysis of all proteins displayed in 1c, showing selected gene ontologies with a positive normalized enrichment score (NES) and adjusted P -value (FDR) < 0.01. Redundant (similar) pathways were removed for clarity. The top most enriched proteins within each ontology are listed within the histograms. GSEA uses a Kolmogorov-Smirnov test, two tailed, with Benjamini-Hochberg (B-H) False Discovery Rate (FDR) correction applied.

Article Snippet: For immunoblot, the following antibodies were used: 1:1000 Rabbit anti-AKAP11 (Custom R171), 1:1000 Rabbit anti-AKAP11 (Custom R173), 1:1000 Mouse anti-PKA R1α (Thermo Fisher Scientific MA5-24981), 1:1000 Rabbit anti-PKA R1α (CST 5675), 1:1000 Rabbit anti-PKA Cα (CST 4782), 1:1000 Rabbit anti-Iqgap1 (Abcam ab86064), 1:1000 Rabbit anti-Dyrk1a (CST 2771), 1:1000 Rabbit anti-GSK3α (CST 4818), 1:1000 Rabbit anti-GSK3β (CST 9315), 1:1000 Mouse anti-P62 (Abcam ab56416), 1:1000 Rabbit anti-LC3A/B (CST 12741), 1:1000 Rabbit anti-VAPA (Proteintech 15275-1-AP), 1:1000 Rabbit anti-β Catenin (CST 8480), 1:1000 Mouse anti-Protein Phosphatase 1 (Santa Cruz SC-7482), 1:1000 Rabbit anti-Sik2 (CST 6919), 1:1000 Rabbit anti-p-GSK3α S21 / anti-p-GSK3β S9 (CST 8566), 1:1000 Rabbit anti-p-GSK3α / anti-p-GSK3β (CST 5676), 1:1000 Rabbit anti- p-GSK3β S9 (CST 9336), 1:1000 Rabbit anti- p-GluA1 S845 (CST 8084), 1:1000 Rabbit anti- GluA1 (CST 13185), 1:1000 Mouse anti- GluA1 (NeuroMab 75-327), 1:1000 Mouse anti-PSD95 (BioLegend 810301), 1:1000 Rabbit anti-Homer1 (Synaptic Systems 160003), 1:1000 Guinea Pig anti-Synaptophysin 1(Synaptic Systems 101004).

Techniques: Immunoprecipitation, Western Blot, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Elevated synaptic PKA activity and abnormal striatal dopamine signaling in Akap11 mutant mice, a genetic model of schizophrenia and bipolar disorder

doi: 10.1038/s41467-025-66504-2

Figure Lengend Snippet: a Number of phosphoproteins reaching a significance threshold of nominal P < 0.05. Fractions of upregulated and downregulated DAPPs are highlighted in red and blue, respectively. b Volcano plots of 12wk synapse phosphoproteomics data for Akap11 +/- and Akap11 -/- vs. WT controls. Phosphoproteomic measurements are normalized to MS-proteomics measurements from the same samples. Known PKA Cα substrates from PhosphoSitePlus are labeled green. AKAP11 phosphopeptides are omitted from Akap11 -/- plots, for clarity. a,b A moderated two-sample t-test was applied to the datasets to compare WT, Akap11 +/- and Akap11 -/- sample groups. c Motif analysis of peptides flanking the phosphorylation site of P < 0.05 phosphosites in A. n(fg) are the number of foreground peptides, and n(bg) are the number of background peptides. Red lines indicate FDR significance, calculated by log odds enrichment. d PTM-SEA of synapse phosphoproteomics data, using known kinase substrates from PhosphoSitePlus. Black borders indicate FDR significance. e ELISA-based PKA activity measurements comparing total lysate and synapse fractions of cortex (left) and striatum-enriched tissue (right). One Unit (U) is defined by the manufacturer as the quantity of PKA that catalyzes the transfer of 1.0 pmol phosphate from ATP to substrate. Two-way ANOVA with Tukey’s post hoc test. f Immunoblotting and quantification of AKAP11, PKA subunits, p-GluA1 S845 and GluA1 in 12wk cortical total lysate. One-way ANOVA with Tukey’s post hoc test. e,f Data are represented as mean ± SEM. See Supplementary Data for detailed statistical information.

Article Snippet: For immunoblot, the following antibodies were used: 1:1000 Rabbit anti-AKAP11 (Custom R171), 1:1000 Rabbit anti-AKAP11 (Custom R173), 1:1000 Mouse anti-PKA R1α (Thermo Fisher Scientific MA5-24981), 1:1000 Rabbit anti-PKA R1α (CST 5675), 1:1000 Rabbit anti-PKA Cα (CST 4782), 1:1000 Rabbit anti-Iqgap1 (Abcam ab86064), 1:1000 Rabbit anti-Dyrk1a (CST 2771), 1:1000 Rabbit anti-GSK3α (CST 4818), 1:1000 Rabbit anti-GSK3β (CST 9315), 1:1000 Mouse anti-P62 (Abcam ab56416), 1:1000 Rabbit anti-LC3A/B (CST 12741), 1:1000 Rabbit anti-VAPA (Proteintech 15275-1-AP), 1:1000 Rabbit anti-β Catenin (CST 8480), 1:1000 Mouse anti-Protein Phosphatase 1 (Santa Cruz SC-7482), 1:1000 Rabbit anti-Sik2 (CST 6919), 1:1000 Rabbit anti-p-GSK3α S21 / anti-p-GSK3β S9 (CST 8566), 1:1000 Rabbit anti-p-GSK3α / anti-p-GSK3β (CST 5676), 1:1000 Rabbit anti- p-GSK3β S9 (CST 9336), 1:1000 Rabbit anti- p-GluA1 S845 (CST 8084), 1:1000 Rabbit anti- GluA1 (CST 13185), 1:1000 Mouse anti- GluA1 (NeuroMab 75-327), 1:1000 Mouse anti-PSD95 (BioLegend 810301), 1:1000 Rabbit anti-Homer1 (Synaptic Systems 160003), 1:1000 Guinea Pig anti-Synaptophysin 1(Synaptic Systems 101004).

Techniques: Phospho-proteomics, Labeling, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot