anti p glua1  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Bipolar and schizophrenia risk gene AKAP11 encodes an autophagy receptor coupling the regulation of PKA kinase network homeostasis to synaptic transmission

doi: 10.1038/s41467-025-66356-w

Figure Lengend Snippet: a Volcano plot of the differentially abundant phosphoprotein sites (DAPPSs) detected by mass-spectrometry in Akap11 -cKO mouse brains. A positive score indicates enrichment, a negative score indicates depletion. The y axis represents statistical confidence FDR for each x axis point. Enriched proteins, defined by FDR < 0.05 and |Log2FC | > 2 SD, are colored in red. FDR was calculated with the moderated two-sided t -test in the LIMMA package for multiple comparisons. b DisGeNET annotations of the DEPPSs through ToppGene in ( a ) ( P -value < 0.05, one-sided hypergeometric test). c Volcano plot of the top 400 predicted PKA phosphorylation sites (normalized by the total protein level) detected by mass-spectrometry in Akap11 -cKO mouse brains. A positive score indicates enrichment, a negative score indicates depletion. The y axis represents statistical confidence FDR for each x axis point. Enriched proteins, defined by FDR < 0.05 and |Log2FC | > 2 SD, are colored in red. FDR was calculated with the moderated two-sided t -test in the LIMMA package for multiple comparisons. d Immunoblotting using antibodies against RIβ, PKA-Cα, GluA1, and AKAP11 in cytosolic and synaptosome fractions and quantification of the results. Data are presented as mean ± SEM ( n = 3 per genotype). Statistical significance was performed using an unpaired two-tailed t -test (cytosolic RIβ: p = 0.0008; cytosolic PKA-Cα: p = 0.0011; synaptosomal RIβ: p = 0.0026; synaptosomal PKA-Cα: p = 0.0108). Each experiment was independently repeated three times with similar results. e Bar graphs of the ELISA results measuring PKA activity in the cytosol and synaptic fractions from Akap11 F /F and Akap11 F/F ; Syn-cre mice. Data are presented as mean ± SEM. Statistical analysis using an unpaired t -test. ( n = 3 per genotype). Statistical significance was performed using an unpaired two-tailed t -test (cytosolic, p = 0.0001; synaptosome, p = 0.0385). Each experiment was independently repeated three times with similar results. f Immunoblotting using antibodies against p-GluA1 S845 and GluA1 in synaptosome and quantification of the results. Data are presented as mean ± SEM ( n = 3 per genotype). Statistical significance was performed using an unpaired two-tailed t -test (p = 0.0014). Each experiment was independently repeated three times with similar results. g Gene Ontology (GO) annotations of the predicted PKA substrates, displaying the top ten enriched pathways ( P -value < 0.05, one-sided hypergeometric test). h Sunburst plots depicting SynGO Biological Process pathways with a two-sided t -test P -value < 0.05. For selected ontologies, representative proteins from predicted PKA substrates within the ontology are displayed.

Article Snippet: Primary antibodies, Rabbit anti-AKAP11 (LS-Bio, LS-C374339), Rabbit anti-RIα (Cell signaling, 5675S), Sheep anti-RIβ (R&D, AF4177), Mouse anti-RIIα (BD, 612242), Rabbit anti-PKACα (Cell signaling, 4782S), Rabbit anti-p-PKACα T197 (Cell signaling, 5661S), Mouse anti-β-actin (Cell signaling, 3700S), Rabbit anti-ADRA2A (Proteintech, 14266-1-AP), Rabbit anti-SPHKAP (Thermo, PA5-27581), Rabbit anti-Synaptophysin (Thermo, MA5-14532), Rabbit anti-GSK3α/β (Cell signaling, 5676S), Rabbit anti-p-GSK3α/β (S21/S9) (Cell signaling, 9331S), Rabbit anti-p-GluA1 S845 (Cell signaling, 8084), Rabbit anti-GluA1 (Cell signaling, 13185), Rabbit anti-Akt1 (Cell signaling, 4691S), Rabbit anti-p-Akt1 S473 (Cell signaling, 4060S), Chicken anti-GFP (Invitrogen, A10262), Rabbit anti-VAPA (Proteintech, 15275-1-AP), Rabbit anti-VAPB (Proteintech, 14477-1-AP), Guinea pig anti-p62 (PROGEN, GP62-C), Rabbit anti-LC3A/B (Cell signaling, 12742S), were diluted in blocking buffer with 0.2% Tween-20 and incubated overnight at 4 °C.

Techniques: Mass Spectrometry, Phospho-proteomics, Western Blot, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Activity Assay