Journal: JCI insight

Article Title: Inhibition of phosphodiesterase 4D suppresses mTORC1 signaling and pancreatic cancer growth.

doi: 10.1172/jci.insight.158098

Figure Lengend Snippet: Figure 2. PDE4D promotes mTORC1 activity through Raptor Ser791 phosphorylation. (A and B) PDE4D inhibits Raptor Ser791 phosphorylation. (A) HA-tagged Raptor was coexpressed with FLAG-tagged PDE4D4 in HEK293A cells for 48 hours; cells were then treated with forskolin (10 μM) for 1 hour. Lysates were immunoprecipitated (IP) with anti-HA antibody and immunoblotted with anti-pPKA substrate (RRXS/T) antibody. (B) Same as A but with FLAG-tagged PDE4D6. (C) PDE4D controls Raptor Ser791 phosphorylation. FLAG-tagged PDE4D (WT), FLAG-tagged PDE4D catalytically inactive mutant (D620A), and HA-tagged Raptor were overexpressed. Cells were treated with forskolin (10 μM) for 1 hour. Lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-pPKA substrate antibody. (D) PDE4D depletion enhances Raptor Ser791 phosphorylation. Cells with PDE4D shRNA were stimulated with forskolin (10 μM) for 1 hour. Lysates were immunoprecipitated with anti-Raptor antibody and immunoblotted for pPKA substrate. Arrow indicates PDE4D. (E) Chemical inhibition of PDE4D decreases mTORC1 activity. HEK293A cells were stimulated with rolipram, roflumilast, and GEBR- 7b for 2 hours and mTORC1 activity was analyzed. (F and G) Raptor Ser791 phosphorylation controls mTORC1 activity. (F) HEK293A cells expressing FLAG- tagged Raptor (WT) or FLAG-tagged Raptor S791A (phospho-defective) were treated with or without GEBR-7b (20 μg/mL) for 2 hours. mTORC1 activity was analyzed. (G) HA-tagged Raptor (WT) or HA-tagged Raptor S791A (phospho-defective) was overexpressed in PDE4D-depleted cells (shPDE4D). mTORC1 activity was analyzed. (H) PDE4D controls mTORC1 activity. Cells were transfected with FLAG-tagged PDE4D4 for 48 hours and stimulated with roflumi- last (50 μM) for 2 hours. mTORC1 activity was analyzed. (I and J) Pharmacologic inhibition of PDE4D enhances Raptor Ser791 phosphorylation. HEK293A cells were pretreated with either roflumilast (50 μM) (I) or GEBR-7b (20 μg/m) (J) for 1 hour and then treated with forskolin (10 μM) for 1 hour. Lysates were immunoprecipitated with anti-Raptor antibody and Raptor Ser791 phosphorylation was assessed. Immunoblots probed for Raptor, pCREB, CREB, FLAG, HA, S6K1, and β-actin are controls. WCL, whole-cell lysates.

Article Snippet: Antibodies against the following proteins were used: β-actin (catalog 3700), HA tag (catalog 3724), pULK1 (catalog 6888), ULK1 (catalog 8054), pS6K1 (catalog 9234), S6K1 (catalog 9202), p4EBP1 (catalog 2855), 4EBP1 (catalog 9452), pCREB (catalog 9198), CREB (catalog 9197), mTOR (catalog 2972), mLST8 (catalog 3274), PKA Catα (catalog 4782), PKA RIα (catalog 5675), pPKA substrate (RRXS/T; catalog 9621) (all Cell Signaling Technology); Raptor (Cell Signaling Technology, catalog 2280 and Bethyl Laboratories, catalog A300-553A), AKAP13 (Thermo Fisher Scientific, catalog PA554078), PKA RIIα (Bethyl Laboratories, catalog A301-670A-M), FLAG tag (Sigma-Aldrich, catalog F1804-50UG), PDE4A (Abcam, catalog ab200383), PDE4C (Proteintech, catalog 21754-1-AP), PDE4B (Thermo Fisher Scientific, catalog 40-1400), and PDE4D (Sigma-Aldrich, catalog ABS22; Bethyl Laboratories, catalog A303-744A; Proteintech, catalog 12918-1-AP; and Thermo Fisher Scientific, catalog PA5-21590).

Techniques: Activity Assay, Phospho-proteomics, Immunoprecipitation, Mutagenesis, shRNA, Inhibition, Expressing, Transfection, Western Blot

Journal: Frontiers in Oncology

Article Title: Identification and Functional Characterization of Anti-metastasis and Anti-angiogenic Activities of Triethylene Glycol Derivatives

doi: 10.3389/fonc.2018.00552

Figure Lengend Snippet: Effect of TD-10 and TD-11 (0.005%; 48 h) on proteins involved in cell migration, apoptosis and proliferation. ( A,B) Immunostaining and immunoblotting of A549 cells treated with TEG derivatives. Downregulation of proteins involved in cell migration and EMT (mortalin, MMP2, vimentin, VEGF, β-catenin, SMAD2/3) was recorded. Downregulation of phosphorylated RB and E2F-1, Cyclin D1, and CDK4 signified moderate inhibition of cell proliferation and migration. Changes in p53, BCL2, Caspase 9 expression were mild and signified lack of apoptosis. ( C,D) Mortalin and VEGF ELISA revealed downregulation in both TD-10 and TD-11 treated cells. Statistical analysis is depicted as * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Fixed cells were incubated with primary antibodies against β-catenin (Santa Cruz, sc-7963), BCL2 (Santa Cruz, sc-492), Caspase 9 (Santa Cruz, sc-7885), E2F-1 (Santa Cruz, sc-251), MMP2 (Santa Cruz, sc-10736), Mortalin , p53 (Santa Cruz, sc-6243), pSMAD 2/3 (Cell Signaling Technologies, 8828S), RB (Cell Signaling Technologies, 9309S), SMAD 2/3 (Santa Cruz, sc-133098), Vimentin (Santa Cruz, sc-5565), VEGF (Santa Cruz, sc-507), Cyclin D1 (Santa Cruz, sc-20044), and CDK4 (Santa Cruz, sc-260) proteins overnight, washed with PBS-PBST-PBS (5 min each), incubated with either Alexa-Fluor 488 goat anti-mouse IgG (Life Technologies, A11029) or Alexa-Fluor 594 goat anti-rabbit IgG (Life Technologies, A11037), depending upon the source of the primary antibodies, for 2 h, washed with PBS-PBST-PBS (5 min each), incubated with Hoechst 33342 stain (Invitrogen Molecular Probes, H3570) for 10 min, washed with PBST-PBS-ultrapure water (5 min each), and mounted on glass slides.

Techniques: Migration, Immunostaining, Western Blot, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Endocrinology

Article Title: Thyroid hormone-induced cell proliferation in GC cells is mediated by changes in G1 cyclin/cyclin-dependent kinase levels and activity.

doi: 10.1210/endo.140.11.7145

Figure Lengend Snippet: FIG. 2. T3 stimulates cyclin E-associated kinase activity. Cell ex- tracts were prepared at the indicated times after culturing of GC cells in the presence or absence of T3. Cyclin E immunoprecipitates were assayed for kinase activity in the presence of histone H1 and [g-32P]ATP as described in Materials and Methods. Kinase reactions were subjected to SDS-PAGE followed by autoradiography. Results shown are representative of two independent experiments. A, Auto- radiograms of cyclin E-cdk phosphorylation of histone H1. B, Quan- titative analysis of the results shown in (A). The results were quan- titated with a PhosphorImager and values expressed as cyclin E-associated kinase activity of cells cultured in the presence of T3 relative to that of cells cultured in the absence of T3. Kinase activity obtained by performing the experiment in the absence of anti-cyclin E antibodies was substracted from its corresponding experimental group. Each time point represents the kinase activity from four in- dividual dishes. Data are expressed as mean 6 SD n 5 8. Statistical significance between the different groups was shown by ANOVA followed by Fisher’s PLSD (P , 0.05).

Article Snippet: Immunoprecipitation, in vitro kinase assay, and Western blot analysis For immunoprecipitation, either 400 mg (H1 phosphorylation) or 100 mg (GST-Rb phosphorylation) of protein extract were incubated with 20 ml of Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 C. The proteins binding nonspecifically to protein A/G Plus-Agarose were pelleted by centrifugation at 4,000 3 g for 5 min and the supernatants incubated for 3 h on ice with 4 mg of anti-cyclin E (sc-481) or anti-cyclin D1 (sc-450) for histone H1 or GST-Rb phosphorylation, respectively.

Techniques: Activity Assay, SDS Page, Autoradiography, Phospho-proteomics, Cell Culture

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: Acta Biochimica et Biophysica Sinica

Article Title: Galangin mitigates glucocorticoid-induced osteoporosis by activating autophagy of BMSCs via triggering the PKA/CREB signaling pathway

doi: 10.3724/abbs.2023063

Figure Lengend Snippet: GAL activated the PKA/CREB-mediated autophagy pathway in Dex-treated human BMSCs (A) Western blot analysis was used to detect the protein expressions of p-CREB, p-PKA, and p-mTOR, and the relative gray values are shown in the histograms (B). (C,D) Cells were pretreated with H89 (10 μM) for 24 h and then treated with Dex (1 μM) and GAL (20 μM) in osteogenic medium for 7 days and 14 days. The cells were analyzed for ALP activity and ARS staining. Scale bar: 100 μm. (E) Western blot analysis was used to detect the expressions of Runx2, OCN, beclin-1, and p62/SQSTM1, and the relative gray values are shown in the histograms (F). Data are shown as the mean±SD ( n=3). * P<0.05, *** P<0.001. NS: not significant.

Article Snippet: SDS-PAGE preparation kits (P0012A), 5× SDS-PAGE protein loading buffer (P0286), BCIP/NBT ALP colour development kit (C3206), H89 (S1643), antibodies against p-(Ser/Thr), PKA substrate (AF9621), and anti-GAPDH antibody (AF5009) were the products of Beyotime Biotechnology (Shanghai, China).

Techniques: Western Blot, Activity Assay, Staining