Journal: Journal of Virology

Article Title: Small Hepatitis B Virus Surface Antigen Promotes Hepatic Gluconeogenesis via Enhancing Glucagon/cAMP/Protein Kinase A/CREB Signaling

doi: 10.1128/jvi.01020-22

Figure Lengend Snippet: SHBs elevates cAMP levels by regulating AC. (A) cAMP levels in the liver of LSL-SHBs and LSL-SHBs/Alb-Cre mice fasted for 16 h ( n = 6 to 7 mice/group). (B to E) Effect of AC inhibitor SQ22536 or PDE inhibitor IBMX on cAMP levels (B), glucose production (C), gluconeogenic gene expression (D), CREB phosphorylation, and PKA activity (E) in Ad-SHBs-infected mouse primary hepatocytes and in Huh7-SHBs cells. Cells were treated with SQ22536 (500 μM) or IBMX (500 μM) for 1 h and then with glucagon (100 nM) stimulation or FSK (10 μM) for 15 min (B), 6 h (C), 2 h (D) and 30 min (E). (F) Huh7 cells were transfected with pcDNA 3.1-SHBs-Flag for 48 h, and then co-IP assays were performed to determine the interaction between SHBs and PKA subunits. The immunoprecipitated complexes with anti-Flag antibody were subjected to immunoblotting with PKA Cα, PKA RIα/β, PKA RIIα, or PKA RIIβ antibody. The glycosylated (gp) and nonglycosylated (p) forms of SHBs were indicated. Arrowhead points to phospho-CREB. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

Article Snippet: The primary antibodies used in this studies included anti-Flag (F1804; Sigma-Aldrich), anti-SHBs (DMABT-51328MH; Creative-Diagnostics, Shirley, NY), anti-CREB (9197S; Cell Signaling Technology [CST], Beverly, MA), anti-phospho-CREB (9198S; CST), anti-phospho-PKA substrate (9624S; CST), anti-PKA Cα (4782S; CST), anti-PKA RIα/β (3927; CST), anti-β-Actin (3700; CST), anti-PKA RIIα (MAB8000; R&D Systems, Minneapolis, MN), anti-PKA RIIβ (PAB18374; Abnova Inc., Walnut, CA), and anti-AC1 (LS-C314759; LifeSpan Biosciences, Seattle, WA).

Techniques: Gene Expression, Phospho-proteomics, Activity Assay, Infection, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot

Journal: Reproductive Medicine and Biology

Article Title: Effect of H89 on the meiotic resumption of pig oocytes

doi: 10.1007/s12522-010-0073-2

Figure Lengend Snippet: PKA activity during meiotic resumption of pig oocytes. A Changes in protein kinase A (PKA) activity of pig oocytes during meiotic maturation. COCs and DOs were cultured for various time periods indicated in the lower row. PKA activities of three oocytes from COCs (a, b) or DOs (c, d) per lane were measured by the phosphorylation of PKA substrate peptide as the substrate. A PKA peptide inhibitor (PKI) was added to the reaction mixture (b, d) to determine the specificity of PKA activity. One result of three separate experiments is shown here. B Localization of active PKA catalytic subunit in pig oocytes from COCs. Immunofluorescent staining was performed on oocytes with anti‐phospho‐PKA catalytic subunit (Thr197) antibody followed by Alexa Fluor 488‐labeled secondary antibody (green) at 0 h (a–c) and after culture for 27 h (d–f). DAPI staining marks chromatin in blue. Scale bar 30 μm

Article Snippet: Kinase activity assay PKA substrate peptide (DLDVPIPGRFDRRVSVAAE [Ala97]‐RII (81–99); Biomol International, Plymouth Meeting, PA, USA) was used as a substrate to determine the activity of PKA by in vitro kinase activity assay of oocytes from COCs and DOs.

Techniques: Activity Assay, Cell Culture, Phospho-proteomics, Staining, Labeling

Journal: Reproductive Medicine and Biology

Article Title: Effect of H89 on the meiotic resumption of pig oocytes

doi: 10.1007/s12522-010-0073-2

Figure Lengend Snippet: Time‐related changes of phosphorylated proteins at serine and threonine residues in pig oocytes from COCs (A) and DOs (B). Sixty oocytes per lane were collected at 0 h and after culture at every time period (3–27 h) given in the lower row and Western blotted with anti‐phospho serine/threonine (anti‐pS/pT) PKA substrate antibody followed by horseradish peroxidase‐conjugated secondary antibody. Each blot represents one of three replicates. C Localization of phosphorylated proteins at serine and threonine residues by PKA in pig oocytes from COCs. Immunofluorescent staining was performed on oocytes with anti‐pS/pT PKA substrate antibody followed by Alexa Fluor 488‐labeled secondary antibody (green) at 0 h (a–c) and after culture for 27 h (d–f). DAPI staining marks chromatin in blue. Scale bar 30 μm

Article Snippet: Kinase activity assay PKA substrate peptide (DLDVPIPGRFDRRVSVAAE [Ala97]‐RII (81–99); Biomol International, Plymouth Meeting, PA, USA) was used as a substrate to determine the activity of PKA by in vitro kinase activity assay of oocytes from COCs and DOs.

Techniques: Western Blot, Staining, Labeling

Journal: Cell Death & Disease

Article Title: Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis

doi: 10.1038/cddis.2013.39

Figure Lengend Snippet: PKA isoenzyme II is dispensable for cAMP-mediated protection against DNR-induced NB4 cell death. ( a ) Immunoblot analysis showing the effect of knockdown of the RII α and RII β subunit of PKA in NB4 RIIKD cells, as compared with NB4 cells transduced with empty vector (NB4 V0 cells). Note that the decline of RII subunits in the NB4 RIIKD cells is compensated by induction of RI α in the NB4 RIIKD cells. ( b and c ) Immunofluorescence analysis showing the sub-cellular localization of the RI ( b ) and RII ( c ) subunits of PKA in NB4 V0 and NB4 RIIKD cells. Note the peri-nuclear position of the RII subunits. DAPI is used to visualize nuclear chromatin. ( d ) NB4 V0 and NB4 RIIKD cells were treated with DNR (5 μ M, 5 h) alone or with N 6 -Bz-cAMP (0.4 mM), and cell death was scored. The data are given as mean±S.E.M., n =3

Article Snippet: Proteins were detected with antibodies against, P-Ser155Bad (murine, corresponds to human P-Ser118), Mcl-1, BAX, Pro-caspase 3, PKA-RII α (Santa Cruz, Biotechnology, Dallas, TX, USA), Bad, Bcl-2, PKA-RI α , PKA-RII β (BD Biosciences), P-Ser112Bad (murine, corresponds to human P-Ser75; Cell Signaling, Danvers, MA, USA), p23 (mouse monoclonal JJ3, generously supplied by Dr. D. O. Toft, Mayo Clinic, Rochester, MN, USA) or β -actin (Amersham Biosciences, Piscataway, NJ, USA).

Techniques: Western Blot, Transduction, Plasmid Preparation, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis

doi: 10.1038/cddis.2013.39

Figure Lengend Snippet: The relative role of PKA isoenzyme I and II to protect against DNR-induced NB4 cell death. ( a and b ) NB4 cells with intact PKA isozymes (NB4 V0 ; a ) or with knocked-down RII subunits (NB4 RIIKD ; b ) were incubated for 4 h with DNR (5 μ M) and various concentrations of 2-Cl-8-HA-cAMP in the absence (•, ) or presence (○,▵) of N 6 -Bz-8-PIP-cAMP (70 μ M), which cooperates with 2-Cl-8-HA-cAMP to activate PKA-I, but not PKA-II. The percentage of dead cells dropped more rapidly when 2-Cl-8-HA-cAMP was combined with N 6 -Bz-8-PIP-cAMP (horizontal arrows), indicating that selective activation of PKA-I could protect both NB4 V0 and NB4 RIIKD cells against DNR. To better assess the protective synergy between the two analogs (horizontal arrow) the initial cell death was set to 100%. The data represent the average of two experiments, and the original data are shown in ,b. Similar results were obtained with other PKA-I synergizing analog combinations (data not shown). The enhanced protection with N 6 -Bz-8-PIP-cAMP was judged by Wilcoxon paired comparison test for the 2-Cl-8-HA-cAMP concentration range 30–600 μ M, and was found significant ( P <0.01) both for the NB4 V0 and the NB4 RIIKD cells. ( c ) NB4 V0 cells were treated as described in a , except that the concentration of Sp-5,6-DCl-cBIMPS was varied in the absence (●) or presence (○) of N 6 -MBC-cAMP (8.5 μ M), in order to test for PKA-II synergism. The data represent the average of two experiments . Similar results (no synergy) were obtained with other PKA-II synergizing analog combinations (data not shown). ( d ) NB4 WT cells were treated for 4 h with DNR (5 μ M) and increasing concentrations of N 6 -Bz-cAMP in the absence (▪) or presence (□) of the PKA-I antagonist R p-8-Br-cAMPS (1 mM, added 0.5 h before DNR). The data are given as mean±S.E.M., n =3. Note that much more N 6 -Bz-cAMP is required to protect against death in the presence of R p-8-Br-cAMPS. The significance of the pairwise difference (± R p-analog at each concentration of N 6 -Bz-cAMP) was evaluated by Student t -test: ** P <0.01, * P <0.05

Article Snippet: Proteins were detected with antibodies against, P-Ser155Bad (murine, corresponds to human P-Ser118), Mcl-1, BAX, Pro-caspase 3, PKA-RII α (Santa Cruz, Biotechnology, Dallas, TX, USA), Bad, Bcl-2, PKA-RI α , PKA-RII β (BD Biosciences), P-Ser112Bad (murine, corresponds to human P-Ser75; Cell Signaling, Danvers, MA, USA), p23 (mouse monoclonal JJ3, generously supplied by Dr. D. O. Toft, Mayo Clinic, Rochester, MN, USA) or β -actin (Amersham Biosciences, Piscataway, NJ, USA).

Techniques: Incubation, Activation Assay, Concentration Assay