Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: GluR1 and neuroinflammation in ACC neurons are involved in CPTP. (A-F) Representative blots and columnar statistical charts show GluR1, TNF-α, and IL-1β levels in contralateral ACC at different time points in Thoracotomy pain (A-C) or Thoracotomy no pain group (D-F), * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs Sham group, the data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 5∼7 rats in each group. (G-I) The photographs of the double immunofluorescence staining show that GluR1, TNF-α, and IL-1β are only colocalized with neuron markers (NeuN) but not with astrocyte marker (GFAP) and microglia marker (Iba1) in the Sham and CPTP groups. The white arrowheads indicate the co-immunostaining: scale bar, 100 μm. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GFAP, glial fibrillary acidic protein; GluR1, glutamate receptor 1; Iba1, ionized calcium-binding adapter molecule 1; IL-1β, interleukin-1β; NeuN, neuron-specific nuclear protein; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Double Immunofluorescence Staining, Marker, Immunostaining, Binding Assay

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: Knockdown of KIBRA in contralateral ACC prevents the upregulation of KIBRA, p -PKMζ/GluR1 signaling pathways, and neuroinflammation in CPTP rats. (A-F) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, TNF-α, and IL-1β levels in ACC analyzed 21 days after the sham operation, thoracotomy, or KIBRA − + thoracotomy. The data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4∼8 rats in each group. ACC, anterior cingulate gyrus; CPTP, chronic post-thoracotomy pain; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Knockdown, Protein-Protein interactions

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: Overexpression of KIBRA in ACC causes allodynia and activates p -PKMζ/GluR1 signaling pathways and neuroinflammation. (A) Experiment designs are shown. (B) Mechanical hyperalgesia ratio on 7, 14, and 21 days after thoracotomy or KIBRA overexpression. **** P < 0.0001 vs Thoracotomy pain group; the data were analyzed by Fisher exact test. (C and D) The threshold% and mechanical pain threshold at different time points for pain rats after injection of rAAV-CMV-wwc1-3xFLAG-WPREs or thoracotomy, * P < 0.05, *** P < 0.001, and **** P < 0.0001 vs Sham group, &&&& P < 0.00001 vs KIBRA + pain group, the data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons test, n = 8 in Sham, n = 13 in Thoracotomy pain, n = 12 in KIBRA + pain, n = 8 in KIBRA + no pain group. (E-J) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, GluR1, TNF-α, and IL-1β levels in ACC analyzed 21 days in Sham, KIBRA + pain, and KIBRA + no pain rats. The data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4 in each group. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Over Expression, Protein-Protein interactions, Injection

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: ACC overexpression KIBRA combined with thoracotomy induced CPTP in all rats. (A) Experiment designs are shown. (B) Incidence of pain on 7, 14, and 21 days after thoracotomy in KIBRA overexpression rats. ** P < 0.01 vs Thoracotomy pain group; the data were analyzed by Fisher exact test. (C and D) The threshold% and mechanical pain threshold at different time points in KIBRA + no pain + Thoracotomy pain, Thoracotomy pain, or Sham group, **** P < 0.0001 vs Sham group, & P < 0.05 vs Thoracotomy pain group. The data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons test, n = 8 in Sham, n = 13 in Thoracotomy pain, n = 14 in KIBRA + no pain + Thoracotomy pain group. (E-J) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, GluR1, TNF-α, and IL-1β levels in ACC analyzed on POD21 in Sham, Thoracotomy pain, or KIBRA + no pain + Thoracotomy pain rats, n = 4 rats in each group. The data were analyzed by 1-way ANOVA followed by Tukey post hoc test. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Over Expression

Journal: IBRO Neuroscience Reports

Article Title: Oxytocin enhances neurogenesis and synaptic plasticity to attenuate age-related cognitive decline in aged mice

doi: 10.1016/j.ibneur.2025.10.008

Figure Lengend Snippet: Intraperitoneal oxytocin injection improves age-related memory impairment in aged mice. (a) Experimental schedule: Twelve-month-old mice were intraperitoneally injected with either saline or oxytocin (0.5 mg/kg, 100 μL) five times per week for 13 weeks. (b) OPR test results: The discrimination index (DI) was calculated as (Time spent in novel place - Time spent in old place) / (Time spent in novel place + Time spent in old place). Oxytocin treatment significantly increased DI compared with saline controls (n = 5 control, n = 6 oxytocin; Mann-Whitney U test, **p < 0.01). (c) DCX immunostaining: Representative immunohistochemical staining of DCX-positive cells in the subgranular zone of the dentate gyrus. Both images shown at same magnification (scale bar: 100 μm). Arrows indicate representative examples of DCX + cells; quantification was based on all DCX + cells within the defined region of interest. The graph shows DCX + cell density (cells/mm²), quantified from three coronal sections per animal (−1.34 to −2.92 mm from bregma, right hemisphere) by a blinded investigator, and presented as the mean±SEM (control group: n = 5, oxytocin group: n = 6). Oxytocin-treated mice exhibited a significant increase in DCX + cell density compared with controls (Mann-Whitney U test, *p < 0.05). (d) Western blotting: Protein expression levels of GluR1, NMDAR2B, and β-actin in the hippocampus were analyzed by Western blotting. Representative blots showing bands at expected molecular weights: GluR1 (∼106 kDa), NMDAR2B (∼180 kDa), β-actin (∼42 kDa). Relative protein expression levels of GluR1 and NMDAR2B were quantified using ImageJ software with background subtraction and normalized to β-actin as loading control. Results, presented as mean±SEM (control group: n = 5, oxytocin group: n = 7), showed significant increase in both protein expression levels in oxytocin-treated mice compared with controls (Mann-Whitney U test, *p < 0.05).

Article Snippet: Equal amounts of protein were separated by 10 % SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK) at 100 V for 90 min. After blocking with 5 % non-fat milk in Tris-buffered saline with 0.1 % Tween 20, the membranes were incubated overnight at 4 °C with primary antibodies against GluR1 (sc-55509, Santa Cruz Biotechnology, Dallas, TX, USA, 1:200), NMDAR2B (AB1557P, Millipore Sigma, Burlington, MA, USA, 1:1000), and β-actin (MA5–11869, Thermo Fisher Scientific, Waltham, MA, USA, 1:1000).

Techniques: Injection, Saline, Control, MANN-WHITNEY, Immunostaining, Immunohistochemical staining, Staining, Western Blot, Expressing, Software

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