Journal: Journal of Virology

Article Title: Begomovirus capsid proteins interact with cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase of its whitefly vector and modulate virus retention within its vector

doi: 10.1128/jvi.02172-24

Figure Lengend Snippet: Primers used in this study ^a

Article Snippet: The PDE4 protein expression was measured by western blot as mentioned earlier; 30 μg total protein was loaded in each lane of 12% polyacrylamide gel, and the PDE4 level was detected using 1:5,000 dilution of a polyclonal anti-PDE4 antibody generated by immunizing two rabbits with a 243 amino acid long partial PDE4 protein (NDESVLENHHLVVEFKLLQKEGCDIFINLSKKQKQTLRKMVIDMVLSTDMSKHMSLLADLKAMVETKKVAGSGVLLLDNYTDRIQVLENLVHCADLSNPTKPLPLYRRWVDLLMEEFFQQGDKEREQNLDISPMCDRHSATIEKSQVGFIDYIVHPLWETWADLVHPDAQEILDMLEENRDWYQSMIPPSPPVNEGENRLDSDVEEGEESEPPNPNPPPVPQDSSIRFQVTLEEGDEDSTAQM) expressed in Escherichia coli (CUSABIO, Houston, Texas, USA).

Techniques: Sequencing, Amplification, Cloning

Journal: Journal of Virology

Article Title: Begomovirus capsid proteins interact with cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase of its whitefly vector and modulate virus retention within its vector

doi: 10.1128/jvi.02172-24

Figure Lengend Snippet: Interaction of PDE4 with CP proteins of CuLCrV, SiGMV, TYLCV, and empty pGBKT7 by one-to-one Y2H mating assays on ( A ) stringent −ALTH and ( B ) less stringent −LTH minimal medium supplemented with α-Xgal.

Article Snippet: The PDE4 protein expression was measured by western blot as mentioned earlier; 30 μg total protein was loaded in each lane of 12% polyacrylamide gel, and the PDE4 level was detected using 1:5,000 dilution of a polyclonal anti-PDE4 antibody generated by immunizing two rabbits with a 243 amino acid long partial PDE4 protein (NDESVLENHHLVVEFKLLQKEGCDIFINLSKKQKQTLRKMVIDMVLSTDMSKHMSLLADLKAMVETKKVAGSGVLLLDNYTDRIQVLENLVHCADLSNPTKPLPLYRRWVDLLMEEFFQQGDKEREQNLDISPMCDRHSATIEKSQVGFIDYIVHPLWETWADLVHPDAQEILDMLEENRDWYQSMIPPSPPVNEGENRLDSDVEEGEESEPPNPNPPPVPQDSSIRFQVTLEEGDEDSTAQM) expressed in Escherichia coli (CUSABIO, Houston, Texas, USA).

Techniques:

Journal: Journal of Virology

Article Title: Begomovirus capsid proteins interact with cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase of its whitefly vector and modulate virus retention within its vector

doi: 10.1128/jvi.02172-24

Figure Lengend Snippet: Validation of interactions between begomovirus CPs and PDE4 by pull-down assay of B. tabaci PDE4 protein from bacterial lysates expressing 6x-his-PDE4 by using GST-fused CPs of TYLCV, CuLCrV, and SiGMV as baits. Mixture of bacterial lysates expressing the CP and PDE4 was used as samples, and only PDE4 protein was used as positive control for detection. Monoclonal anti-GST and anti-6x-his antibodies were used to detect CPs and PDE4, respectively. The ponceau-stained membrane shows the protein amounts of the individual lanes.

Article Snippet: The PDE4 protein expression was measured by western blot as mentioned earlier; 30 μg total protein was loaded in each lane of 12% polyacrylamide gel, and the PDE4 level was detected using 1:5,000 dilution of a polyclonal anti-PDE4 antibody generated by immunizing two rabbits with a 243 amino acid long partial PDE4 protein (NDESVLENHHLVVEFKLLQKEGCDIFINLSKKQKQTLRKMVIDMVLSTDMSKHMSLLADLKAMVETKKVAGSGVLLLDNYTDRIQVLENLVHCADLSNPTKPLPLYRRWVDLLMEEFFQQGDKEREQNLDISPMCDRHSATIEKSQVGFIDYIVHPLWETWADLVHPDAQEILDMLEENRDWYQSMIPPSPPVNEGENRLDSDVEEGEESEPPNPNPPPVPQDSSIRFQVTLEEGDEDSTAQM) expressed in Escherichia coli (CUSABIO, Houston, Texas, USA).

Techniques: Biomarker Discovery, Pull Down Assay, Expressing, Positive Control, Staining, Membrane

Journal: Journal of Virology

Article Title: Begomovirus capsid proteins interact with cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase of its whitefly vector and modulate virus retention within its vector

doi: 10.1128/jvi.02172-24

Figure Lengend Snippet: Localization of PDE4 protein and TYLCV in the midgut of ( A ) viruliferous and ( B ) non-viruliferous B. tabaci reared on TYLCV-infected and non-host cotton plants, respectively. TYLCV was detected using polyclonal anti-TYLCV CP antibody and cy5 (green) conjugated secondary antibody. PDE4 was detected using polyclonal anti-PDE4 antibody and cy3 (red) conjugated secondary antibody. The overlay of green and red channels is indicated in yellow, and midgut cell nuclei, stained with DAPI, are indicated in blue. The gut area magnified to 60× and 180× are depicted in white square boxes.

Article Snippet: The PDE4 protein expression was measured by western blot as mentioned earlier; 30 μg total protein was loaded in each lane of 12% polyacrylamide gel, and the PDE4 level was detected using 1:5,000 dilution of a polyclonal anti-PDE4 antibody generated by immunizing two rabbits with a 243 amino acid long partial PDE4 protein (NDESVLENHHLVVEFKLLQKEGCDIFINLSKKQKQTLRKMVIDMVLSTDMSKHMSLLADLKAMVETKKVAGSGVLLLDNYTDRIQVLENLVHCADLSNPTKPLPLYRRWVDLLMEEFFQQGDKEREQNLDISPMCDRHSATIEKSQVGFIDYIVHPLWETWADLVHPDAQEILDMLEENRDWYQSMIPPSPPVNEGENRLDSDVEEGEESEPPNPNPPPVPQDSSIRFQVTLEEGDEDSTAQM) expressed in Escherichia coli (CUSABIO, Houston, Texas, USA).

Techniques: Infection, Staining

Journal: Journal of Virology

Article Title: Begomovirus capsid proteins interact with cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase of its whitefly vector and modulate virus retention within its vector

doi: 10.1128/jvi.02172-24

Figure Lengend Snippet: Relative quantitation of PDE4 mRNA in viruliferous whitefly adults post 12, 24, and 48 hours of AAP on ( A ) CuLCrV- or ( B ) TYLCV-infected squash/tomato plants compared with non-viruliferous whitefly (NV) adults exposed to non-infected plants. ( C ) Abundance of PDE4 protein detected by western blot analysis of soluble proteins extracted from viruliferous whitefly adults post 12 hours of virus acquisition (CuLCrV/TYLCV) compared with non-viruliferous (NV) adults. PDE4 band volumes (intensity) normalized to α-tubulin protein bands of viruliferous (CuLCrV/TYLCV) and NV control samples are depicted in bar graphs. ( D ) Quantitation of cAMP (picomoles per milligram whitefly protein) in viruliferous (post 12 hours of CuLCrV acquisition) and non-viruliferous (NV) whitefly adults.

Article Snippet: The PDE4 protein expression was measured by western blot as mentioned earlier; 30 μg total protein was loaded in each lane of 12% polyacrylamide gel, and the PDE4 level was detected using 1:5,000 dilution of a polyclonal anti-PDE4 antibody generated by immunizing two rabbits with a 243 amino acid long partial PDE4 protein (NDESVLENHHLVVEFKLLQKEGCDIFINLSKKQKQTLRKMVIDMVLSTDMSKHMSLLADLKAMVETKKVAGSGVLLLDNYTDRIQVLENLVHCADLSNPTKPLPLYRRWVDLLMEEFFQQGDKEREQNLDISPMCDRHSATIEKSQVGFIDYIVHPLWETWADLVHPDAQEILDMLEENRDWYQSMIPPSPPVNEGENRLDSDVEEGEESEPPNPNPPPVPQDSSIRFQVTLEEGDEDSTAQM) expressed in Escherichia coli (CUSABIO, Houston, Texas, USA).

Techniques: Quantitation Assay, Infection, Western Blot, Virus, Control

Journal: Journal of Virology

Article Title: Begomovirus capsid proteins interact with cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase of its whitefly vector and modulate virus retention within its vector

doi: 10.1128/jvi.02172-24

Figure Lengend Snippet: ( A ) Quantitation of cAMP (picomoles per milligram whitefly protein) in B. tabaci adults fed for 48 hours on 20% sucrose diet with either 0.8% ethanol (control) or 200 µM rolipram, an inhibitor of PDE4. ( B ) Relative quantitation of CuLCrV DNA and ( C ) TYLCV DNA retained (normalized to the β-tubulin gene of the whitefly) in whitefly adults post virus acquisition (24 hours) from infected squash/tomato plants and overnight gut clearing when pre-fed on 20% sucrose diet (48 hours) with 0.8% ethanol (control) or with 200 µM rolipram.

Article Snippet: The PDE4 protein expression was measured by western blot as mentioned earlier; 30 μg total protein was loaded in each lane of 12% polyacrylamide gel, and the PDE4 level was detected using 1:5,000 dilution of a polyclonal anti-PDE4 antibody generated by immunizing two rabbits with a 243 amino acid long partial PDE4 protein (NDESVLENHHLVVEFKLLQKEGCDIFINLSKKQKQTLRKMVIDMVLSTDMSKHMSLLADLKAMVETKKVAGSGVLLLDNYTDRIQVLENLVHCADLSNPTKPLPLYRRWVDLLMEEFFQQGDKEREQNLDISPMCDRHSATIEKSQVGFIDYIVHPLWETWADLVHPDAQEILDMLEENRDWYQSMIPPSPPVNEGENRLDSDVEEGEESEPPNPNPPPVPQDSSIRFQVTLEEGDEDSTAQM) expressed in Escherichia coli (CUSABIO, Houston, Texas, USA).

Techniques: Quantitation Assay, Control, Virus, Infection

Journal: Journal of Virology

Article Title: Begomovirus capsid proteins interact with cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase of its whitefly vector and modulate virus retention within its vector

doi: 10.1128/jvi.02172-24

Figure Lengend Snippet: Relative quantitation of mRNA (normalized to the β-tubulin gene of the whitefly) of ( A ) PDE4 or ( B ) AC in F1 B. tabaci adults reared on tomato plants transformed with PDE4_TRV2 or AC_TRV2 constructs compared with TRV2 control plants.

Article Snippet: The PDE4 protein expression was measured by western blot as mentioned earlier; 30 μg total protein was loaded in each lane of 12% polyacrylamide gel, and the PDE4 level was detected using 1:5,000 dilution of a polyclonal anti-PDE4 antibody generated by immunizing two rabbits with a 243 amino acid long partial PDE4 protein (NDESVLENHHLVVEFKLLQKEGCDIFINLSKKQKQTLRKMVIDMVLSTDMSKHMSLLADLKAMVETKKVAGSGVLLLDNYTDRIQVLENLVHCADLSNPTKPLPLYRRWVDLLMEEFFQQGDKEREQNLDISPMCDRHSATIEKSQVGFIDYIVHPLWETWADLVHPDAQEILDMLEENRDWYQSMIPPSPPVNEGENRLDSDVEEGEESEPPNPNPPPVPQDSSIRFQVTLEEGDEDSTAQM) expressed in Escherichia coli (CUSABIO, Houston, Texas, USA).

Techniques: Quantitation Assay, Transformation Assay, Construct, Control

Journal: Journal of Virology

Article Title: Begomovirus capsid proteins interact with cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase of its whitefly vector and modulate virus retention within its vector

doi: 10.1128/jvi.02172-24

Figure Lengend Snippet: Relative quantitation of ( A ) CuLCrV and ( B ) TYLCV DNA retained (normalized to the β-tubulin gene of the whitefly) in F1 B. tabaci adults reared on either PDE4_TRV2 or TRV2 plants post 24 hours of virus acquisition from infected plants followed by overnight gut clearing on cotton plants. ( C ) Relative quantitation of CuLCrV DNA retained post 24 hours of virus acquisition and overnight gut clearing in F1 adults reared on AC_TRV2 compared to TRV2 control.

Article Snippet: The PDE4 protein expression was measured by western blot as mentioned earlier; 30 μg total protein was loaded in each lane of 12% polyacrylamide gel, and the PDE4 level was detected using 1:5,000 dilution of a polyclonal anti-PDE4 antibody generated by immunizing two rabbits with a 243 amino acid long partial PDE4 protein (NDESVLENHHLVVEFKLLQKEGCDIFINLSKKQKQTLRKMVIDMVLSTDMSKHMSLLADLKAMVETKKVAGSGVLLLDNYTDRIQVLENLVHCADLSNPTKPLPLYRRWVDLLMEEFFQQGDKEREQNLDISPMCDRHSATIEKSQVGFIDYIVHPLWETWADLVHPDAQEILDMLEENRDWYQSMIPPSPPVNEGENRLDSDVEEGEESEPPNPNPPPVPQDSSIRFQVTLEEGDEDSTAQM) expressed in Escherichia coli (CUSABIO, Houston, Texas, USA).

Techniques: Quantitation Assay, Virus, Infection, Control

Journal: Cellular signalling

Article Title: Phosphodiesterase 4 in inflammatory diseases: Effects of apremilast in psoriatic blood and in dermal myofibroblasts through the PDE4/CD271 complex.

doi: 10.1016/j.cellsig.2016.01.007

Figure Lengend Snippet: Fig. 3. PDE4/CD271 expression in healthy and pathologic dermis. (A) PDE4A, PDE4B, PDE4C, PDE4D isoforms and CD271 in dermis from healthy controls and patients with psoriasis, AD, and DLE were detected by IHC. Fast red was used as chromogen. Bar (shown in top left image = 200 μm) (B) The expression intensity of PDE4A, PDE4B, PDE4C, PDE4D isoforms and CD271 in (A) was quantitatively measured using ImageJ software as described in the Materials and Methods. Relative staining intensities are shown, normalized to intensities in healthy dermis for each individual antibody.

Article Snippet: The blots were blocked for 2 h in blocking buffer (PBS buffer, pH 7.4 with 0.2% Tween 20 and 5% nonfat milk) and incubated with rabbit polyclonal anti-human PDE4A antibody (1:1000; Proteintech Group, Chicago, IL), rabbit polyclonal anti-human PDE4B antibody (1:300; Santa Cruz Biotechnology), rabbit polyclonal anti-human PDE4C antibody (1:300; Santa Cruz Biotechnology), rabbit polyclonal anti-human PDE4D antibody (1:300; Santa Cruz Biotechnology) or mouse monoclonal -SMA (1:3000; Sigma-Aldrich) or mouse monoclonal anti-β-actin (1:1000; AC C EP TE D M AN U SC R IP T Sigma) overnight at 4oC.

Techniques: Expressing, Software, Staining

Journal: Cellular signalling

Article Title: Phosphodiesterase 4 in inflammatory diseases: Effects of apremilast in psoriatic blood and in dermal myofibroblasts through the PDE4/CD271 complex.

doi: 10.1016/j.cellsig.2016.01.007

Figure Lengend Snippet: Fig. 4. PDE4/CD271 expression in fibroblasts and myofibroblasts in vitro. (A) α-SMA expression in DF and DM obtained after treatment with diluent (control) or TGF-β1 (1 ng/mL) for 6 days by immunofluorescence in situ. Cell nuclei were counterstained with DAPI (blue); bar = 20 μm. (B) After diluent (control) or TGF-β1 treatment, levels of α-SMA were determined by Western blot analysis. Vinculin was used as a loading control. (C) Levels of PDE4A, PDE4B, PDE4C, and PDE4D mRNA in DF and DM were determined by RT-PCR analysis. Bar graph shows the average densitometry values normalized to β-actin. (D) PDE4 isoform expression in DF and DM was determined by Western blot analysis. β-Actin was used as a loading control. Bar graphs show the average densitometry values normalized to β-actin (fold-expression). (E) DF and DM protein extracts were immunoprecipitated with CD271 antibody and immunoblotted with PDE4A, PDE4B, PDE4C, and PDE4D antibodies and with PAS-only as control.

Article Snippet: The blots were blocked for 2 h in blocking buffer (PBS buffer, pH 7.4 with 0.2% Tween 20 and 5% nonfat milk) and incubated with rabbit polyclonal anti-human PDE4A antibody (1:1000; Proteintech Group, Chicago, IL), rabbit polyclonal anti-human PDE4B antibody (1:300; Santa Cruz Biotechnology), rabbit polyclonal anti-human PDE4C antibody (1:300; Santa Cruz Biotechnology), rabbit polyclonal anti-human PDE4D antibody (1:300; Santa Cruz Biotechnology) or mouse monoclonal -SMA (1:3000; Sigma-Aldrich) or mouse monoclonal anti-β-actin (1:1000; AC C EP TE D M AN U SC R IP T Sigma) overnight at 4oC.

Techniques: Expressing, In Vitro, Control, In Situ, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

Journal:

Article Title: Specific Role of Neuronal Nitric-oxide Synthase when Tethered to the Plasma Membrane Calcium Pump in Regulating the ?-Adrenergic Signal in the Myocardium * S⃞

doi: 10.1074/jbc.M809112200

Figure Lengend Snippet: A, shown is an image of nNOS co-localization to PMCA4b. Isolated adult mouse cardiomyocytes (upper panel) and neonatal rat cardiomyocytes (lower panel) were co-stained with anti-PMCA4 and anti-nNOS antibodies. B, nNOS-specific activity and total NOS activity were significantly reduced in heart extracts from PMCA4b-TG mice compared with wild type littermates (n = 8; *, p < 0.05). C, cGMP levels were also significantly decreased in heart homogenates from PMCA4b-TG mice compared with wild type controls (n = 8; *, p < 0.05). However, cAMP levels (D) as well as PKA activity (E) were significantly increased in PMCA4b-TG mice versus wild type (n = 8; *, p < 0.05 versus WT). F, Western blot analyses of sGC and PKA in the heart extracts are shown. G, a quantification of band density after normalization with α-tubulin expression suggested that the expression levels of these proteins were not altered in PMCA4b-TG mice (n = 8).

Article Snippet: Primary antibodies used for the Western blot analysis were as follows: polyclonal anti-PMCA4 (SWANT); monoclonal anti-PMCA (5F10), anti-soluble guanylyl cyclase (sGC), anti-protein kinase A (PKA), anti-cardiac troponin I, anti-phospho-Ser 22 /Ser 23 troponin I, anti-PDE2, anti-PDE3, and anti-PDE4 (Abcam); anti-phospholamban and anti-phospho-Ser 16 phospholamban (Upstate); and anti-nNOS (Affinity Bioreagents).

Techniques: Isolation, Staining, Activity Assay, Western Blot, Expressing

Journal: Arthritis Research & Therapy

Article Title: Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors

doi: 10.1186/s13075-015-0771-6

Figure Lengend Snippet: Apremilast inhibits lipopolysaccharide ( LPS )-induced TNF-α release via cyclic adenosine monophosphalphate (cAMP). a Western blot of PDE4 was performed in the Raw 264.7 cell line. b Raw 264.7 cells were incubated with increasing concentrations of apremilast 30 minutes before incubation with or without LPS 1 μM during 20 minutes. Intracellular cAMP levels were then measured as described under “ ”. c Western blot of p-cAMP responsive element binding protein ( p-CREB ) and CREB shows that apremilast promotes CREB phosphorylation after incubation with LPS 1 μg/ml for 30 minutes. d Cumulative concentration response curves to apremilast (6 nM−1 μM) were performed in the Raw 264.7 cells 30 minutes before incubation with LPS 1 μM during 4 h. IC 50 values were determined as described under “Materials and methods”. Data represent means ± standard error of the mean of four independent experiments. Statistical analysis was performed by two-way ANOVA: apremilast *** P <0.001, LPS: not significant. PDE4 phosphodiesterase 4,

Article Snippet: Membranes where incubated overnight (4 °C) with primary rabbit polyclonal anti-pCREB (Abcam, Cambridge, MA, USA), mouse monoclonal anti-CREB (Abcam), rabbit polyclonal anti-PDE4 (Abcam) and mouse monoclonal anti-Actin (1:1000 each).

Techniques: Western Blot, Incubation, Binding Assay, Concentration Assay