pka  (Cell Signaling Technology Inc)

Journal: Poultry Science

Article Title: PKA and PKC signaling pathways mediate vitamin D₃-regulated intestinal phosphorus absorption in broiler chickens

doi: 10.1016/j.psj.2026.106762

Figure Lengend Snippet: Effects of dietary vitamin D 3 supplementation on jejunal phosphorylation levels of PKA, PKC, PI3K, p38MAPK, and ERK in broiler chickens (19 d) (Experiment 1). The control and vitamin D 3 diets contained 0 and 1000 IU/kg vitamin D 3 , respectively. (a) p-PKA/t-PKA; (b) p-PKC/t-PKC; (c) p-PI3K/t-PI3K; (d) p-p38MAPK/t-p38MAPK; (e) p-ERK/t-ERK. Protein abundance values are means ± SD of four replicates per treatment (1 broiler per replicate) (n = 4). Values with different letters differ between treatments ( P < 0.05).

Article Snippet: PVDF membranes were incubated with primary antibodies against NaPi-IIb (1:1000, A9460; ABclonal, Wuhan, China), phosphorylated PKA (p-PKA; 1:1000, 5661S), phosphorylated PI3K (p-PI3K; 1:1000, 4228), phosphorylated ERK (p-ERK; 1:1000, 9101S), phosphorylated p38MAPK (p-p38MAPK; 1:1000, 9216S), total PKA (t-PKA; 1:1000, 5842S) (Cell Signaling Technology, Boston, USA), total PI3K (t-PI3K; 1:10000, 60225-1-Ig), total ERK (t-ERK; 1:8000, 11257-1-AP-50), total p38MAPK(t-p38MAPK; 1:1000, 14064-1-AP), total PKC (t-PKC; 1:1000, 21991-1-AP), phosphorylated PKC (p-PKC; 1:5000, 29123-1-AP), and GAPDH (1:10000, 60004-1-Ig) (Proteintech, Wuhan, China).

Techniques: Phospho-proteomics, Control, Quantitative Proteomics

Journal: Poultry Science

Article Title: PKA and PKC signaling pathways mediate vitamin D₃-regulated intestinal phosphorus absorption in broiler chickens

doi: 10.1016/j.psj.2026.106762

Figure Lengend Snippet: Effects of the PKA inhibitor H-89 on jejunal phosphorylation levels of PKC, PI3K, p38MAPK, and ERK in broiler chickens (10–15 d) (Experiment 2). (a) p-PKC/t-PKC; (b) p-PI3K/t-PI3K; (c) p-p38MAPK/t-p38MAPK; (d) p-ERK/t-ERK. Protein abundance values are means ± SD of four replicates per treatment (1 broiler per replicate) (n = 4). Values with different letters differ between treatments ( P < 0.05).

Article Snippet: PVDF membranes were incubated with primary antibodies against NaPi-IIb (1:1000, A9460; ABclonal, Wuhan, China), phosphorylated PKA (p-PKA; 1:1000, 5661S), phosphorylated PI3K (p-PI3K; 1:1000, 4228), phosphorylated ERK (p-ERK; 1:1000, 9101S), phosphorylated p38MAPK (p-p38MAPK; 1:1000, 9216S), total PKA (t-PKA; 1:1000, 5842S) (Cell Signaling Technology, Boston, USA), total PI3K (t-PI3K; 1:10000, 60225-1-Ig), total ERK (t-ERK; 1:8000, 11257-1-AP-50), total p38MAPK(t-p38MAPK; 1:1000, 14064-1-AP), total PKC (t-PKC; 1:1000, 21991-1-AP), phosphorylated PKC (p-PKC; 1:5000, 29123-1-AP), and GAPDH (1:10000, 60004-1-Ig) (Proteintech, Wuhan, China).

Techniques: Phospho-proteomics, Quantitative Proteomics

Journal: Poultry Science

Article Title: PKA and PKC signaling pathways mediate vitamin D₃-regulated intestinal phosphorus absorption in broiler chickens

doi: 10.1016/j.psj.2026.106762

Figure Lengend Snippet: Effects of the PKC inhibitor staurosporine on duodenal phosphorylation levels of PKA, PI3K, p38MAPK, and ERK in broiler chickens (13 d) (Experiment 3). (a) p-PKA/t-PKA; (b) p-PI3K/t-PI3K; (c) p-p38MAPK/t-p38MAPK; (d) p-ERK/t-ERK. Protein abundance values are means ± SD of four replicates per treatment (1 broiler per replicate) (n = 4). Values with different letters differ between treatments ( P < 0.05).

Article Snippet: PVDF membranes were incubated with primary antibodies against NaPi-IIb (1:1000, A9460; ABclonal, Wuhan, China), phosphorylated PKA (p-PKA; 1:1000, 5661S), phosphorylated PI3K (p-PI3K; 1:1000, 4228), phosphorylated ERK (p-ERK; 1:1000, 9101S), phosphorylated p38MAPK (p-p38MAPK; 1:1000, 9216S), total PKA (t-PKA; 1:1000, 5842S) (Cell Signaling Technology, Boston, USA), total PI3K (t-PI3K; 1:10000, 60225-1-Ig), total ERK (t-ERK; 1:8000, 11257-1-AP-50), total p38MAPK(t-p38MAPK; 1:1000, 14064-1-AP), total PKC (t-PKC; 1:1000, 21991-1-AP), phosphorylated PKC (p-PKC; 1:5000, 29123-1-AP), and GAPDH (1:10000, 60004-1-Ig) (Proteintech, Wuhan, China).

Techniques: Phospho-proteomics, Quantitative Proteomics

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Heatmap showing relative expression of COX2 and type I ISGs in mice treated with either vehicle (Veh) or doxorubicin (Doxo) (GEO: GSE223698). (B) Heatmap showing relative expression of COX2 and type I ISGs in proliferating cells (Prof) and senescence cells (Sen) (GEO: GSE196610). (C) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for either 24 or 48 hours. Cell lysates were subjected to RT-qPCR to assess mRNA levels of COX2 (n = 3). (D) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for either 24 or 48 hours in the presence or absence of 1 µM celecoxib (COX2i). Cell lysates were collected and analyzed by ELISA to measure extracellular PGE 2 levels (n = 3). (E) A schematic illustrates PGE 2 -cAMP-PKA signaling. (F) THP-1 macrophages were treated with 1 µM PGE 2 in the presence or absence of 1 µM EP4 inhibitor (EP4i) for 16 hours. Cell lysates were collected and analyzed by ELISA to measure intracellular cAMP levels (n = 3). (G) THP-1 macrophages were treated with 1 µM PGE 2 at indicated concentrations in the presence or absence of 1 µM EP4 inhibitor (EP4i) for 16 hours. Cell lysates were collected and subjected to immunoblotting with the indicated antibodies. (H) THP-1 macrophages expressing ISRE-Luciferase were mock-infected or infected with HSV-1 (MOI = 1) in the presence of either 1 µM PGE 2 or 1 µM forskolin and 50 µM IBMX. At 16 h.p.i, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (I-J) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 24 h.p.i, cell lysates were subjected to RT-qPCR to assess mRNA levels of IFNβ (n = 3) (I) , and cell supernatants were subjected to ELISA to measure secreted levels of IFNβ (n = 3) (J) . (K) THP-1 macrophages expressing ISRE-Luciferase were mock-infected or infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 16 h.p.i, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (L) THP-1 macrophages were infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 16 h.p.i, cell lysates were collected and subjected to RT-qPCR to assess HSV-1 UL30 genomic abundance (n = 3). Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. P -values are indicated.

Article Snippet: Antibodies for PKA substrates (#9624), PKA Cα (#5842), PKA RIα/β (#3927), GAPDH (#2118), STING (#13647), pTBK1 S172 (5483), TBK1 (#3504), pIRF3 S396 (#4947), IRF3 (#4302), TFAM (#8076), HSP90 (#4877), pUB S65 (#62802), COX4 (#4850), pPINK1 S228 (#89010), STOML2 (#89199), Vinculin (#13901) were purchased from Cell Signaling Technology.

Techniques: Expressing, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Reporter Assay, Activity Assay

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Proteomic workflow in HEK293 cells with doxycycline-inducible expression of PKA Cα wild-type (WT) or W197R mutant (MUT). (B) The top 8 significantly enriched KEGG pathways within the mitochondrial quality control category identified from the proteomic dataset. (C) Schematic of the mt-mKeima mitophagy reporter. (D) Representative Airyscan live-cell imaging of THP-1 macrophages expressing mt-mKeima sensor treated with 1 µM PGE 2 in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi) for 16 hours. Scale bar, 10 µm. (E) Quantification of mitolysosome numbers shown in (D) (n = 10). Data were quantified from one representative experiment of three. (F) Representative images of live-cell 4D lattice light sheet imaging on THP-1 macrophages treated as in (D) . Scale bar, 20 µm. (G-H) Quantification of net mitolysosome displacement (n = 10) (G) and average mitolysosome speed (n = 12) (H) from imaging in (F) . (I) THP-1 macrophages treated with PGE 2 at indicated concentrations in the presence or absence of 1 µM PKA inhibitor (PKAi) for 16 hours. Mitochondrial fractions were isolated and subjected to immunoblotting with indicated antibodies. Band intensities were quantified and normalized to COX4 expression for PINK1 (J) and pUB Ser65 (K) (n = 3). (L) Working model illustrating that PGE 2 induces mitophagy and mitochondrial biogenesis to enhance mitochondrial homeostasis in a EP4- and PKA-dependent manner. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. p -values are indicated.

Article Snippet: Antibodies for PKA substrates (#9624), PKA Cα (#5842), PKA RIα/β (#3927), GAPDH (#2118), STING (#13647), pTBK1 S172 (5483), TBK1 (#3504), pIRF3 S396 (#4947), IRF3 (#4302), TFAM (#8076), HSP90 (#4877), pUB S65 (#62802), COX4 (#4850), pPINK1 S228 (#89010), STOML2 (#89199), Vinculin (#13901) were purchased from Cell Signaling Technology.

Techniques: Expressing, Mutagenesis, Control, Live Cell Imaging, Imaging, Isolation, Western Blot

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Network of mitochondrial quality control proteins interacting with wild-type (WT) and mutant (MUT) PKA Cα with BFDR σ; 0.2. Interactors are colored by log 2 fold change (WT/MUT spectral counts) with WT-specific interactors in dark purple and MUT-specific interactors in dark green. Edges to the central bait (yellow) represent interactions detected in this study. (B) Whole cell lysates of THP-1 macrophages were subjected to pulldown using 8-AHA-cAMP (RIα), Rp-8-AHA-cAMPS (holoenzyme), or HaloLink resin (control), followed by immunoblotting with indicated antibodies. (C-D) THP-1 macrophages were stimulated with 1 µM PGE 2 for 6 hours, followed by incubation with 1 µM cycloheximide for the indicated times. Cell lysates were collected and subjected to immunoblotting with the indicated antibodies. Representative blots from three independent experiments are shown (C) . Band intensities for STOML2 were quantified and normalized to HSP90 intensity (n = 3) (D) . (E-G) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or STOML2 siRNA (siSTOML2), followed by treatment with PGE 2 at indicated concentrations for 16 hours. Mitochondrial fractions were isolated and subjected to immunoblotting with the indicated antibodies (E) . Band intensities for PINK1 (F) and pUB Ser65 (G) were quantified and normalized to COX4 intensity (n = 3). (H) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or STOML2 siRNA (siSTOML2), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, cytosol fractions were isolated and subjected to qPCR to assess the presence of mt-Dloop and mt-ND1 regions (n=3). (I) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or STOML2 siRNA (siSTOML2), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, whole cell lysates were collected and subjected to RT-qPCR to assess mRNA levels of IFNβ (n=3). (J) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or STOML2 siRNA (siSTOML2), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, whole cell lysates were collected and subjected to qPCR to assess HSV-1 UL30 genomic abundance (n=3). All experiments were performed with three independent biological replicates and repeated at least twice with reproducible results. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. p -values are indicated.

Article Snippet: Antibodies for PKA substrates (#9624), PKA Cα (#5842), PKA RIα/β (#3927), GAPDH (#2118), STING (#13647), pTBK1 S172 (5483), TBK1 (#3504), pIRF3 S396 (#4947), IRF3 (#4302), TFAM (#8076), HSP90 (#4877), pUB S65 (#62802), COX4 (#4850), pPINK1 S228 (#89010), STOML2 (#89199), Vinculin (#13901) were purchased from Cell Signaling Technology.

Techniques: Control, Mutagenesis, Western Blot, Incubation, Transfection, Isolation, Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) HEK293 cells expressing STOML2-3xFlag were treated with 1 µM forskolin and 50 µM IBMX for the indicated times. Whole cell lysates were subjected to immunoprecipitation using DYKDDDDK Fab-Trap agarose and immunoblotting with the indicated antibodies. (B) Top: domain architecture of STOML2 and predicted PKA phosphorylation sites (Scansite 4.0; PhosphoSitePlus). Bottom: ClustalW alignment showing evolutionary conservation surrounding STOML2 Ser29. (C) Representative structural model of the interaction between the active pocket of PKA Cα and STOML2 N-terminal (aa 18 to 37) (ipTM = 0.9). Gray: PKA Cα, green: PKA Cα residue D167, yellow: STOML2, magenta: STOML2 residue S29. (D) HEK293 cells expressing either STOML2 wild-type (WT) or S29A mutant (MUT) were treated with 1 µM forskolin and 50 µM IBMX for 6 hours. Cell lysates were subjected to immunoprecipitation using DYKDDDDK Fab-Trap agarose and immunoblotting with the indicated antibodies. (E) Schematic illustrating the generation of THP-1 macrophages expressing either STOML2 wild-type (WT) or S29A mutant (MUT) via lentivirus transduction. (F) THP-1 macrophages expressing either STOML2 wild-type (WT) or S29A mutant (MUT) were stimulated with 1 µM PGE 2 for 16 hours. Cell lysates were subjected to immunoprecipitation using DYKDDDDK Fab-Trap agarose and immunoblotting with the indicated antibodies. (G) Representative Airyscan live-cell imaging of THP-1 macrophages expressing either STOML2 wild-type (WT) or S29A mutant (MUT) together with the mt-mKeima sensor were stimulated with 1 µM PGE 2 for 16 hours. Scale bar, 10 µm. (H) Quantification of mitolysosome numbers from (G) (n = 10). (I) THP-1 macrophages expressing either STOML2 wild-type (WT) or S29A mutant (MUT) were stimulated with 1 µM PGE 2 for 16 hours. Mitochondrial fractions were collected and subjected to immunoblotting with indicated antibodies. (J) A schematic illustrating that PKA activation promotes STOML2 phosphorylation at Ser29 which subsequently induces PINK1-mediated mitophagy in response to PGE 2 stimulation. All experiments were performed with three independent biological replicates and repeated at least twice with reproducible results. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. p -values are indicated.

Article Snippet: Antibodies for PKA substrates (#9624), PKA Cα (#5842), PKA RIα/β (#3927), GAPDH (#2118), STING (#13647), pTBK1 S172 (5483), TBK1 (#3504), pIRF3 S396 (#4947), IRF3 (#4302), TFAM (#8076), HSP90 (#4877), pUB S65 (#62802), COX4 (#4850), pPINK1 S228 (#89010), STOML2 (#89199), Vinculin (#13901) were purchased from Cell Signaling Technology.

Techniques: Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Residue, Mutagenesis, Transduction, Live Cell Imaging, Activation Assay