Journal: Cellular and Molecular Immunology

Article Title: IFN-α production by human mononuclear cells infected with varicella-zoster virus through TLR9-dependent and -independent pathways

doi: 10.1038/cmi.2010.84

Figure Lengend Snippet: PKR but not DNA-PK was involved in IFN-α production by PBMCs upon VZV infection. (a) PBMCs were pre-incubated with and without DMNB or 2AP for 30 min and then cocultured with VZV. Supernatants were collected 48 h after stimulation and subjected to detection of IFN-α. The IFN-α production of PBMCs induced by VZV could be partially inhibited by 2AP, but not by DMNB. Data presented were calculated from six replicated experiments. (b) Proteins of PBMCs collected at different time points after stimulation with and without VZV were subjected to western blot. Total PKR and p-PKR were increased as PBMCs were stimulated with VZV. Plots represent three replicated experiments. DMNB, 4,5-dimethoxy-2-nitrobenzaldehyde; DNA-PK, DNA-dependent protein kinase; IFN, interferon; NC, negative control; ND, non-detectable; PBMC, peripheral blood mononuclear cell; PKR, double-stranded RNA-dependent protein kinase; p-PKR, phospho-PKR; VZV, varicella-zoster virus; 2AP, 2-aminopurine.

Article Snippet: Monoclonal antibodies against human phospho-PKR (p-PKR), total PKR (Cell Signaling Technology, Inc., Beverly, MA, USA) and actin (Zymed Laboratories Inc., South San Francisco, CA, USA) were used as first antibodies to recognize p-PKR, total PKR and actin.

Techniques: Infection, Incubation, Western Blot, Negative Control

Journal: Aging Cell

Article Title: PKR knockout in the 5xFAD model of Alzheimer's disease reveals beneficial effects on spatial memory and brain lesions

doi: 10.1111/acel.12887

Figure Lengend Snippet: PKR knockout modulates cognition and Aβ formation. (a–c) Behavior was studied in 9‐month‐old male and female wild‐type ( n = 17), 5xFAD ( n = 14), PKRKO ( n = 14), and 5xFAD × PKRKO (double mutant, DM, n = 10) mice. (a) In the novel object location test, the percentages of time exploring the displaced object (new location, NL, 24 hr after first exposure) and the unmoved object (old location, OL) were compared. Two‐way ANOVA interaction F (3,51) = 21.06, p < 0.0001, OL vs. NL, Holm–Sidak's test, p < 0.0001 for wild‐type, nonsignificant for 5xFAD, p < 0.0001 for PKRKO, and p < 0.0001 for DM. (b) Performances in the starmaze. S1, S2 = session number (5 trials per session). Repeated measures ANOVA, genotype effect p = 0.0184, sessions effect p < 0.0001, interaction F (7,100) = 18.96, post hoc Holm–Sidak's test. (c) Anxiety assessment in the elevated plus maze. Time in open arm between 5xFAD and double‐mutant mice: Kruskal–Wallis test p < 0.0001, post hoc Mann–Whitney test. (d) PET scan analysis of amyloid burden in PKRKO, 5xFAD, and DM mice ( n = 4–6 per group). All results are expressed as the standard uptake value ratio (SUVr) relative to the cerebellum. (e, f) Quantification of Aβ levels in the hippocampus of 30‐week‐old mice by immunoblot analysis (e) with GAPDH used as the loading control, and by ELISA analysis (f) ( n = 6 mice per group). Kruskal–Wallis, p < 0.0001, Dunn's post hoc test. (g) BACE1 levels in the hippocampus of 30‐week‐old mice ( n = 10 mice per group). Kruskal–Wallis, p = 0.0011, Dunn's post hoc test. (I) Aβ immunohistochemistry with Moab antibody, revealed with horseradish peroxidase, in the subiculum of 30‐week‐old mice ( n = 5 mice per group). Significant reduction of Aβ load in double‐mutant mice after quantification. Mann–Whitney test, p = 0.016. (a–h) Data are means ± SEM . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Antibodies for Aβ (Mouse monoclonal 3G5; Thermo Fisher, Waltham MA, USA), total PKR (rabbit polyclonal antibody 18,244–1‐AP 1/1,000; Proteintech, Rosemont, IL, USA), cleaved caspase 3 (rabbit monoclonal, 5A1E, Cell Signaling), phospho‐bcl2 (mouse monoclonal antibody C‐2, Santa Cruz Biotechnology) and BAX (rabbit monoclonal antibody D2E11, Cell Signaling) were used to carry out western blotting according to the suppliers' instructions.

Techniques: Knock-Out, Mutagenesis, MANN-WHITNEY, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Aging Cell

Article Title: PKR knockout in the 5xFAD model of Alzheimer's disease reveals beneficial effects on spatial memory and brain lesions

doi: 10.1111/acel.12887

Figure Lengend Snippet: PKR knockout enhances neuroprotection in 5xFAD mice. (a) Caspase 3 activity in the hippocampus of 30‐week‐old wild‐type, 5xFAD, PKRKO, and 5xFAD×PKRKO (double mutant, DM) mice ( n = 10 mice per group). Two‐way ANOVA, F (3,36) = 3.718, p = 0.0025; Tukey's post hoc test. (b–d). Immunoblotting analysis of caspase 3 (b), phospho‐bcl2 (c), and BAX (d) in the hippocampus of 30‐week‐old mice ( n = 6 mice per group). GAPDH was used as the loading control. (b) One‐way ANOVA, F (3,16) = 1.546, Tukey's post hoc test, p < 0.0001. (c) Kruskal–Wallis, Dunn's post hoc tests, p = 0.001. (d), One‐way ANOVA, F (3,29) = 0.2476, p = 0.0014, Tukey's post hoc test. (e) Fluoro‐jade C fluorescence in the subiculum of 30‐week‐old mice ( n = 6 mice per group) (horizontal bar 50 µM). One‐way ANOVA, F (3,19) = 3.811, p = 0.0001, Tukey's post hoc test. (f) Synaptophysin immunofluorescence in the subiculum of 30‐week‐old wild‐type, 5xFAD, and DM mice ( n = 5 mice per group). (horizontal bar 20 µM) Kruskal–Wallis test, p < 0.0001, Dunn's post hoc test. (a–f) Data are means ± SEM * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Antibodies for Aβ (Mouse monoclonal 3G5; Thermo Fisher, Waltham MA, USA), total PKR (rabbit polyclonal antibody 18,244–1‐AP 1/1,000; Proteintech, Rosemont, IL, USA), cleaved caspase 3 (rabbit monoclonal, 5A1E, Cell Signaling), phospho‐bcl2 (mouse monoclonal antibody C‐2, Santa Cruz Biotechnology) and BAX (rabbit monoclonal antibody D2E11, Cell Signaling) were used to carry out western blotting according to the suppliers' instructions.

Techniques: Knock-Out, Activity Assay, Mutagenesis, Western Blot, Fluorescence, Immunofluorescence

Journal: Aging Cell

Article Title: PKR knockout in the 5xFAD model of Alzheimer's disease reveals beneficial effects on spatial memory and brain lesions

doi: 10.1111/acel.12887

Figure Lengend Snippet: PKR knockout reduces neuroinflammation in 5xFAD mice. (a) IBA1 immunohistochemistry in the subiculum of 30‐week‐old wild‐type, 5xFAD, PKRKO, and 5xFAD×PKRKO (double mutant, DM) mice ( n = 5 mice per group) (horizontal bar 20 µM). Quantitative results show a significant reduction of IAB1 immunostaining in double‐mutant mice as compared to 5xFAD mice. One‐way ANOVA, F (3,16) = 0.5339, p < 0.0001, Tukey's post hoc test. (b–e) Luminex analysis of IL‐1β (b), TNFα (c), IFNγ (d), and IL‐6 (e) levels in the hippocampus of 30‐week‐old wild‐type, 5xFAD, PKRKO, and 5xFAD×PKRKO (double mutant, DM) mice ( n = 6 mice per group). Kruskal–Wallis test, (c) p = 0.0001, (d–e), p < 0.0001, Dunn's post hoc test. (f–h) Neurons and microglia derived from wild‐type or PKRKO mouse embryos, as indicated, were cocultured and treated for 24 hr with 1 µg/ml LPS. Caspase 3 activity was measured in the cell lysates at the end of the treatment ( n = 3–5 cocultures per group). One‐way ANOVA, F (7, 22) = 2.936, p < 0.0001; Tukey's post hoc test. Neurons and microglia from wild‐type or PKRKO embryos were cocultured as in f, or cultured alone as indicated. Cytokine levels were measured using Luminex in the supernatant 1 day after a 24‐hr treatment with vehicle (control) or 1 µg/ml LPS. IL‐1β, TNFα, and IL‐6 (g: cocultures)(h; pure cultures). Two‐way ANOVA, interaction F (3, 27) = 3.935, p = 0.0189; treatment effect F (3, 27) = 4.010, p = 0.0175; and genotype effect F (1, 27) = 22,47, p < 0.0001, respectively; Tukey's post hoc test. (a–i) Data are means ± SEM , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Antibodies for Aβ (Mouse monoclonal 3G5; Thermo Fisher, Waltham MA, USA), total PKR (rabbit polyclonal antibody 18,244–1‐AP 1/1,000; Proteintech, Rosemont, IL, USA), cleaved caspase 3 (rabbit monoclonal, 5A1E, Cell Signaling), phospho‐bcl2 (mouse monoclonal antibody C‐2, Santa Cruz Biotechnology) and BAX (rabbit monoclonal antibody D2E11, Cell Signaling) were used to carry out western blotting according to the suppliers' instructions.

Techniques: Knock-Out, Immunohistochemistry, Mutagenesis, Immunostaining, Luminex, Derivative Assay, Activity Assay, Cell Culture

Journal: Aging Cell

Article Title: PKR knockout in the 5xFAD model of Alzheimer's disease reveals beneficial effects on spatial memory and brain lesions

doi: 10.1111/acel.12887

Figure Lengend Snippet: Signaling pathways in Alzheimer's disease in which PKR is implicated. Schematic representation of the pathways PKR may modulate based on the current and previous findings in neurons and microglia. In microglia, Aβ or LPS can trigger a PKR‐dependent production of cytokines (such as TNFα or IL‐1β) that are secondarily released or a PKR‐dependent activation of NF‐κB leading to inflammation. In neurons, cytokines from microglia or Aβ oligomers can trigger a PKR‐dependent increase in BACE1 activity, ISR activation via eIF2α, and a PKR‐dependent apoptosis via phospho‐bcl2 and BAX

Article Snippet: Antibodies for Aβ (Mouse monoclonal 3G5; Thermo Fisher, Waltham MA, USA), total PKR (rabbit polyclonal antibody 18,244–1‐AP 1/1,000; Proteintech, Rosemont, IL, USA), cleaved caspase 3 (rabbit monoclonal, 5A1E, Cell Signaling), phospho‐bcl2 (mouse monoclonal antibody C‐2, Santa Cruz Biotechnology) and BAX (rabbit monoclonal antibody D2E11, Cell Signaling) were used to carry out western blotting according to the suppliers' instructions.

Techniques: Activation Assay, Activity Assay

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: Hypoxia induces morphological changes and PRK1 activation in PC12 cells. PC12 cells were incubated under normoxic (21% O2; N-co) or hypoxic (1% O2; H-co) conditions. (a) After 6-8h incubation, cells were stained with Hoechst/PI for photomicrographs of viable (blue) and dead (pink) cells. (b) Quantification of the percentage of cell death [pink cells × 100/total cell number]. (c) F-actin–associated cytoskeletal reorganization over 0-24h normoxia/hypoxia in phalloidin-stained cells (green). Dashed lines in panels c(i-v) indicate the area selected for higher magnification in panels c(vi-x). (d) Neurite formation of PC12 cells after incubation under normoxic and hypoxic conditions for 24h. Three to five independent microscopic fields were studied and the percentage of neurite formation was calculated [neurite-bearing cells × 100/total cell number]. Extensions longer than twice the cell diameter were considered neurites. (e) Hypoxic treatment for 5 min increases PRK1 activation compared to normoxic cells. Cell lysates were analyzed by Western blot with anti-phospho-PRK1 and anti-pan-PRK1 antibodies. The intensity was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the ratio (anti-phospho/anti-pan–PRK1) was calculated. Pictures and blots are representative of 3 to 5 independent experiments. Values represent the means ± S.E.M., n=3-5. Differences were analyzed using unpaired one-tailed t-test: (b) ++P<0.01, (d) ++P<0.01 and (e) +++P<0.001.

Article Snippet: Anti-pan-PRK1 was obtained from BD Biosciences (San Jose CA, USA).

Techniques: Activation Assay, Incubation, Staining, Western Blot, One-tailed Test

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: Guanosine and NGF mediate neuroprotective effects and increase PRK1 activation in PC12 cells. Hypoxic cells (H-co) were treated with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or a combination of guanosine and NGF (Guo+NGF). (a) Cell death was quantified with Hoechst/PI after 6-8h treatment as described in experimental procedures. The white line indicates cell death in normoxic control. (b) F-actin–associated cytoskeleton was studied in phalloidin-stained cells (green) 24h after stimulation. Nuclei were labeled with Hoechst 33342 dye (blue). (c) Neurite formation was calculated in hypoxic PC12 cells (H-co), as well as in cells treated with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or a combination of guanosine and NGF (Guo+NGF) for 24h. White lines indicate neurite formation under normoxic condition. (d) PRK1 activation was studied in cell lysates stimulated under normoxic/ hypoxic conditions and with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or guanosine and NGF together (Guo+NGF) for 5 min by immunoprobing the blot with anti-phospho-PRK1 and anti-pan-PRK1 antibodies. White lines indicate PRK1 activation under normoxic condition. The intensity was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the anti-phospho/anti-pan–PRK1 ratio was calculated. White lines indicate PRK1 activation under normoxic condition. Pictures and blots are representative of 4 to 11 independent experiments. Values represent the means ± S.E.M., n=3-11. Differences were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test: (a) *P<0.05, **P<0.01 (c) *P<0.05, ***P<0.001 and (d) *P<0.05, ***P<0.001.

Article Snippet: Anti-pan-PRK1 was obtained from BD Biosciences (San Jose CA, USA).

Techniques: Activation Assay, Staining, Labeling

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: siRNA-mediated PRK1 knockdown is specific and suppresses viability of hypoxic PC12 cells. PC12 cells were transfected with synthetic siRNA for PRK1 or scrambled non-targeting siRNA duplexes. (a) Cell extracts were analyzed 24h to 72h after transfection by immunoblotting for anti-pan-PRK1, anti-pan-PRK2 (specificity control) and anti-pan-AKT (loading control). (b) The intensity of protein bands was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the anti-pan-PRK1/anti-pan-AKT ratio was calculated. (c) The effect of siRNA-mediated knockdown on cell viability was studied after additional stimulation of hypoxic PC12 cells (H-co) with 500μM guanosine (Guo) for 6-8h and staining for Hoechst/PI. (d) The percentage of hypoxic cell death in PRK1 knockdown cells was calculated. Pictures and blots are representative of 3 to 5 independent experiments and values represent the means ± S.E.M., n=3-5. Differences were analyzed using unpaired one-tailed t-test: (b) ++P<0.01, +++P<0.001 and (d) ++P<0.01 (control siRNA vs. PRK1 siRNA).

Article Snippet: Anti-pan-PRK1 was obtained from BD Biosciences (San Jose CA, USA).

Techniques: Transfection, Western Blot, Staining, One-tailed Test

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: siRNA-mediated PRK1 knockdown inhibits guanosine- and NGF-mediated cytoskeletal reorganization, GAP-43 expression, and neurite formation in hypoxic PC12 cells. PC12 cells were transfected with siRNA for PRK1 or control siRNA duplexes. (a) siRNA-transfected hypoxic PC12 cells (H-co) were treated with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or both guanosine and NGF (Guo+NGF) for 24h and then stained with FITC-phalloidin (green); nuclei were labeled with Hoechst 33342 (blue) [panels a(i-iv and ix-xii)]. F-actin accumulation at neurite growth cones and branching points was observed (arrowheads). In parallel, hypoxic neurons (H-co) were immunostained for anti-pan-GAP-43 (red) [panels a(v-viii and xiii-xvi), the inserts in panel a(v-viii) represent normoxic cells] after stimulation with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or guanosine plus NGF (Guo+NGF) for 24h. Condensed levels of GAP-43 in growth cones (asterisk) and in perinuclei (arrows) are shown. Photomicrographs are representative of 3 to 5 independent experiments, each with 4 to 6 independent microscopic fields. (b) GAP-43 expression was investigated by analyzing cell lysates from transfected hypoxic PC12 cells (H-co) and hypoxic cells treated with 500μM guanosine (Guo), 50ng/mL NGF (NGF), or a combination of both (Guo+NGF) for 3 days. Intensity was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the anti-pan-GAP-43/anti-pan-AKT ratio was calculated. (c) The effect of PRK1 knockdown on guanosine- and NGF-mediated neurite formation by stimulation of siRNA-transfected cells with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or guanosine plus NGF (Guo+NGF) under hypoxic conditions for 3 days was calculated as described in experimental procedures. (d) Transfected PC12 cells treated with guanosine (Guo), 5ng/mL NGF (NGF), or guanosine plus NGF (Guo+NGF) under hypoxic conditions for 24h were stained for gamma tubulin (green) and anti-pan-GAP-43 (red) [panels d(i-viii)]. Pictures are representative of 3 independent experiments. Values represent the means ± S.E.M., n=3-9. Differences were analyzed using unpaired one-tailed t-test: (b) +P<0.05, ++P<0.01 and (c) +P<0.05, ++P<0.01 (control siRNA vs. PRK1 siRNA).

Article Snippet: Anti-pan-PRK1 was obtained from BD Biosciences (San Jose CA, USA).

Techniques: Expressing, Transfection, Staining, Labeling, One-tailed Test

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: Guanosine protects cell viability and the neuronal network in hypoxic primary cerebellar granule neurons. Primary cerebellar granule neurons were subjected to normoxia or hypoxia. (a) After 6-8h treatment normoxic (N-co) or hypoxic cells (H-co) were stained for viability with Hoechst/PI according to the protocol in experimental procedures [panels a(i and ii)]. Treatment with 500μM guanosine (Guo) reduced cell death [panels a(ii and iii)]. (b) In phase contrast photomicrographs, neurites of cerebellar granule neurons were visualized [panels b(i and ii)] after 6-8h incubation. Additional 500μM guanosine (Guo) improved stability of the neuronal network in hypoxic cerebellar granule cells [panels b(ii and iii)]. (c) PRK1 activation was studied after 5 min treatment under normoxic conditions (N-co), hypoxic conditions (H-co) and in hypoxic neurons incubated with 500μM guanosine (Guo). Pictures and blots are representative of 6 to 9 independent experiments. Values represent the means ± S.E.M., n=6-9. Differences were analyzed using unpaired one-tailed t-test: (c) +P<0.05.

Article Snippet: Anti-pan-PRK1 was obtained from BD Biosciences (San Jose CA, USA).

Techniques: Staining, Incubation, Activation Assay, One-tailed Test

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: PRK1 is essential for neuroprotection and GAP-43 expression in hypoxic cerebellar granule neurons. Freshly isolated primary cerebellar granule neurons were transfected with control or specific siRNA for PRK1. (a) PRK1 protein expression was studied 48h post-transfection by immunoblotting. The intensity was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the anti-pan-PRK1/anti-pan-AKT ratio was calculated. (b) Transfected cerebellar granule neurons were exposed to hypoxic conditions (H-co) [panels b(i and ii)], treated with 500μM guanosine (Guo) [panels b(iii and iv)] for 6-8h, and stained with Hoechst/PI. (c) Quantification of the effect of PRK1 knockdown on cell death. (d) GAP-43 protein fluorescence intensity was studied by staining normoxic (N-co) [panel d(i)] and hypoxic cerebellar granule neurons (H-co) [panels d(ii and iii)]. Hypoxic cells were also treated with 500μM guanosine (Guo) [panels d(iv and v)]. Pictures and blots are representative of 3 to 4 independent experiments. Values represent the means ± S.E.M., n=4. Differences were analyzed using unpaired one-tailed t-test: (a) +P<0.05 and (c) ++P<0.01.

Article Snippet: Anti-pan-PRK1 was obtained from BD Biosciences (San Jose CA, USA).

Techniques: Expressing, Isolation, Transfection, Western Blot, Staining, Fluorescence, One-tailed Test

Journal: Computational and Structural Biotechnology Journal

Article Title: Biased activation of the vasopressin V2 receptor probed by molecular dynamics simulations, NMR and pharmacological studies

doi: 10.1016/j.csbj.2024.10.039

Figure Lengend Snippet: Pharmacological properties and binding poses of the ligands in V2R . (A) Binding properties (left), effects on cAMP accumulation (central-left) and βarr2 recruitment (center-right and right). TR-FRET was expressed as FRET ratio (665 nm/620 nm x 10,000). * ** *, p < 0.0001; ns, not significant (p > 0.05). (B) Extracted HMQC spectra of K100 2.65 and K116 3.29 resonances of V2R K268 6.32 R bound to par dLVP (sky blue) or par MCF14 (magenta), and reduced by ascorbic acid (mauve taupe and light violet, respectively). Ratios of peak volumes in the paramagnetic and diamagnetic (ligand red ) forms of each agonist are shown to the right. Volumes were quantitated using the nlinLS subroutine of NMRpipe . They identify K100 265 methyl groups as the closest to the nitroxide ion. Error bars come from two experiments recorded with the receptor purified from two different batches. (C) Snapshots of tagged and non-tagged agonists during MD simulations. The nitroxide cage is squared. Tagged and non-tagged agonists showed identical binding poses in the pocket, while the tag and linker swayed around the entrance of the pocket. (D) Probability density distribution of the distances between the PROXYL nitroxide ion and the methyl groups of K100 2.65 (plain line) and K116 3.32 (dashed line) during the MD simulations. Due to inherent inaccuracies in the simulations, the values may not be quantitatively interpreted for comparison with the NMR data. They illustrate a qualitative trend that aligns with the NMR spectra.

Article Snippet: Briefly, 35000 cells in 100 μl DMEM were transfected with 100 ng of total DNA (10 μl NaCl 150 mM containing 5 ng of pRK5-SNAPtag-V2R, 5 ng of either pcDNA3.1-βarr1-RlucII or pcDNA3.1-βarr2-RlucII, 30 ng of pcDNA3.1-rGFP-CAAX and 60 ng of salmon sperm DNA (ThermoFisher)) using transfecting agent polyethylenimine (PEI linear, MW 25KTM, 1 mg/ml, Polysciences) at a ratio of 0.3 μl PEI per 100 ng DNA.

Techniques: Binding Assay, Purification, Comparison

Journal: Cell reports

Article Title: Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production

doi: 10.1016/j.celrep.2019.11.009

Figure Lengend Snippet: (A) Of the 90 phosphosites we observed on metabolic enzymes, PKL pS113 is the most prominent, with greater than 2-fold change in both the HFD-fed compared to chow-fed samples and the HFD-fed compared to the HFD-PKCε ASO. Data are plotted as in Figure S1B except only phosphorylation on metabolic enzymes is shown. Phosphosites <2-fold change in both comparisons are in green, phosphosites >2-fold change in 1 comparison are in blue, and PKL pS113 (>2-fold change in both comparisons) is in red.

Article Snippet: 20 μL PKL total (ProteinTech pAb, or Santa Cruz mAb) and PKL pS113 antibodies were added to 10 μL bed volume protein A/G PLUS-Agarose beads (Santa Cruz), incubated for 1hr at RT and washed 2x with PBS.

Techniques:

Journal: Cell reports

Article Title: Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production

doi: 10.1016/j.celrep.2019.11.009

Figure Lengend Snippet: (A) Primary hepatocytes treated with glucose and the gluconeogenic precursors pyruvate and lactate showed increased PKL pS113 compared to just glucose or glucose with the PKA activator IBMX.

Article Snippet: 20 μL PKL total (ProteinTech pAb, or Santa Cruz mAb) and PKL pS113 antibodies were added to 10 μL bed volume protein A/G PLUS-Agarose beads (Santa Cruz), incubated for 1hr at RT and washed 2x with PBS.

Techniques:

Journal: Cell reports

Article Title: Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production

doi: 10.1016/j.celrep.2019.11.009

Figure Lengend Snippet: (A and B) Rat hepatoma cells (A) and primary hepatocytes (B) were immunostained for PKL pS113 and show strong nuclear localization (4‘,6-diamidino-2-phenylindole [DAPI]) of the pS113 signal. Scale bar represents 50 μm.

Article Snippet: 20 μL PKL total (ProteinTech pAb, or Santa Cruz mAb) and PKL pS113 antibodies were added to 10 μL bed volume protein A/G PLUS-Agarose beads (Santa Cruz), incubated for 1hr at RT and washed 2x with PBS.

Techniques:

Journal: Cell reports

Article Title: Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production

doi: 10.1016/j.celrep.2019.11.009

Figure Lengend Snippet: (A) Rat primary hepatocytes were treated with and without 1 μM CDKi and then immunostained for PKL pS113. DAPI (blue) and PKL pS113 (red); scale bar represents 50 μm.

Article Snippet: 20 μL PKL total (ProteinTech pAb, or Santa Cruz mAb) and PKL pS113 antibodies were added to 10 μL bed volume protein A/G PLUS-Agarose beads (Santa Cruz), incubated for 1hr at RT and washed 2x with PBS.

Techniques: