anti erk1 2 phospho  (Revvity)

Journal: bioRxiv

Article Title: Artificial Intelligence Enables the Label-Free Identification of Chronic Myeloid Leukemia Cells with Mitochondrial Morphological Alterations

doi: 10.1101/2023.07.26.550632

Figure Lengend Snippet: A. ERK1/2 expression and phosphorylation of UT-7/EPO cells with or without BCR-ABL1 transduction. B. Drp1 and ERK1/2 expression and phosphorylation of UT-7/EPO cells with or without BCR-ABL1 transduction after treatments with 0, 1, 10, and 50 µM of the MEK inhibitor U0126 for 4 h. C. Drp1 and ERK1/2 expression and phosphorylation of K562 cells after treatments with 0, 1, 10, and 50 µM of the MEK inhibitor U0126 for 4 h. D. Drp1 and ERK1/2 expression and phosphorylation of KU812 cells after treatments with 0, 1, 10, and 50 µM of the MEK inhibitor U0126 for 4 h. E. Representative confocal laser scanning microscopy images of UT-7/EPO with or without BCR-ABL1 treated with U0126 (10 µM for 24 h) or DMSO (control). Mitochondria are shown in green, and nuclei are shown in blue. F. The proportions of cells with different mitochondrial morphologies in UT-7/EPO with or without BCR-ABL1 treated with U0126 (10 µM for 24 h) or DMSO (control). Among the cells imaged using confocal laser scanning microscopy, the sizes of mitochondria were classified as Elongated, Intermediate, or Fragmented, and their respective ratios were presented. ***p < 0.01 by Fisher’s exact test. BCR-ABL1 (-), BCR-ABL1 -untransduced; BCR-ABL1 (+), BCR-ABL1 -transduced; ERK, extracellular signal-regulated kinase; Drp, dynamin-related protein; Ser, serine; DMSO, dimethyl sulfoxide.

Article Snippet: The following antibodies were employed: anti-DRP1 Ab (Clone: D8H5, Cell Signaling Technology, Danvers, MA); anti-phospho Drp1 (Ser616) Ab (Cell Signaling Technology); anti-Mitofsin1 Ab (Clone: 11E91H12, Abcam, Cambridge, UK); anti-Mitofsin2 Ab (Clone: D2D10, Cell Signaling Technology); anti-OPA1 Ab (Clone: 18/OPA-1, Becton Dickinson); anti-ERK1/2 antibody (clone W15133B, BioLegend); anti-ERK1/2 phospho(Thr202/Tyr204) antibody (clone: 6B8B69, BioLegend); and anti-Actin Ab (clone: 13E5, Cell Signaling Technology).

Techniques: Expressing, Transduction, Confocal Laser Scanning Microscopy, Control

Journal: Pharmaceutics

Article Title: Pistacia vera Extract Potentiates the Effect of Melatonin on Human Melatonin MT 1 and MT 2 Receptors with Functional Selectivity

doi: 10.3390/pharmaceutics15071845

Figure Lengend Snippet: Effect of selected plant extracts on phospho-ERK levels. Detection of ERK1/2 phosphorylation in HEK293 empty vector transfected cells (mock) ( A ) or stably expressing MT 1 ( B ) or MT 2 ( C ) receptors. Extracts were tested at a saturation concentration (3.2 × 10 −4 M i.e., 0.16 mg/mL, except Ex18 , which was tested at 1.07 × 10 −7 M, equivalent to 0.005 mg/mL) and MLT (1 µM). Data are expressed as mean ± SEM, n = 3–5). ( A ) (** p < 0.01; *** p < 0.0001; **** p < 0.0001; One-way ANOVA). ( B ) (* p = 0.02; two-tailed Student’s t -test); ( C ) (* p = 0.01; ** p = 0.004; two-tailed Student’s t -test).

Article Snippet: A quantitative analysis of ERK1/2 activation in non-transfected HEK293 cells (mock) or stably expressing human MT 1 or MT 2 was performed using the p-ERK1/2 AlphaLISA Sure-fire ultra-Assay kit (PerkinElmer, Waltham, MA, USA) as described [ ].

Techniques: Plasmid Preparation, Transfection, Stable Transfection, Expressing, Concentration Assay, Two Tailed Test

Journal: Pharmaceutics

Article Title: Pistacia vera Extract Potentiates the Effect of Melatonin on Human Melatonin MT 1 and MT 2 Receptors with Functional Selectivity

doi: 10.3390/pharmaceutics15071845

Figure Lengend Snippet: Concentration-dependence of the effect of selected plant extracts on phospho-ERK levels. Effect of increasing concentrations of Ex17 , 18 and 19 on phospho-ERK1/2 levels in HEK293 empty vector transfected cells (mock) vs. HEK293 expressing MT 1 ( A , C , E ) or MT 2 receptors ( B , D , F ). Inset represents the effect of MLT and Ex18 at the maximum concentration on mock cells. Data represent the mean of three independent experiments performed in duplicates. ( A , E ) (* p = 0.02; ns: not significant; two-tailed Student’s t -test).

Article Snippet: A quantitative analysis of ERK1/2 activation in non-transfected HEK293 cells (mock) or stably expressing human MT 1 or MT 2 was performed using the p-ERK1/2 AlphaLISA Sure-fire ultra-Assay kit (PerkinElmer, Waltham, MA, USA) as described [ ].

Techniques: Concentration Assay, Plasmid Preparation, Transfection, Expressing, Two Tailed Test

Journal: Pharmaceutics

Article Title: Pistacia vera Extract Potentiates the Effect of Melatonin on Human Melatonin MT 1 and MT 2 Receptors with Functional Selectivity

doi: 10.3390/pharmaceutics15071845

Figure Lengend Snippet: Replotting of experimental data obtained with Ex18 ( Pistacia vera) based on the actual melatonin amount of Ex18 determined using HPLC-MS. ( A , B ) Competitive binding curves, concentration–response curve of the inhibition of cAMP production ( C , D ), ERK1/2 activation ( E , F ) and β-arrestin2 recruitment ( G , H ). Experiments with synthetic melatonin were run in parallel. Data are expressed as mean ± SEM, n = 3. Curves were analysed using non-linear regression. The log IC 50 and EC 50 values were compared between Ex18 and MLT via the extra sum-of-squares F test. p -values for log IC 50 were **** p < 0.0001 ( A ), *** p = 0.0008 ( B ) and for log EC 50 ** p = 0.002 ( C ), ** p = 0.009 ( D ), * p = 0.07 ( E ), * p = 0.02 ( F ). ns, not significant ( G , H ).

Article Snippet: A quantitative analysis of ERK1/2 activation in non-transfected HEK293 cells (mock) or stably expressing human MT 1 or MT 2 was performed using the p-ERK1/2 AlphaLISA Sure-fire ultra-Assay kit (PerkinElmer, Waltham, MA, USA) as described [ ].

Techniques: Binding Assay, Concentration Assay, Inhibition, Activation Assay

Journal: Pharmaceutics

Article Title: Pistacia vera Extract Potentiates the Effect of Melatonin on Human Melatonin MT 1 and MT 2 Receptors with Functional Selectivity

doi: 10.3390/pharmaceutics15071845

Figure Lengend Snippet: Potentiation of melatonin effect by Pistacia vera extract on ERK1/2 activation. Effects of increasing concentrations of MLT, Ex18 alone or in presence of 10 nM MLT are shown on p-ERK levels in HEK293 cells expressing MT 1 ( A ) or MT 2 ( B ) receptors. The concentration–response curves of Ex18 were calculated in molarity after having determined its content in MLT using HPLC-MS. Data are presented as mean ± SEM, n =3–4. Curves were analysed using non-linear regression. The E max and Log EC 50 values were compared between Ex18 vs. Ex18 + MLT (10 nM) using the extra sum-of-squares F test. p -values for E max and log EC 50 were p = 0.17, 0.47 for MT 1 ( A ), respectively.

Article Snippet: A quantitative analysis of ERK1/2 activation in non-transfected HEK293 cells (mock) or stably expressing human MT 1 or MT 2 was performed using the p-ERK1/2 AlphaLISA Sure-fire ultra-Assay kit (PerkinElmer, Waltham, MA, USA) as described [ ].

Techniques: Activation Assay, Expressing, Concentration Assay

Journal: iScience

Article Title: A 2B adenosine receptor activation and modulation by protein kinase C

doi: 10.1016/j.isci.2023.107178

Figure Lengend Snippet:

Article Snippet: AlphaScreen cAMP kit, SureFire®p-ERK1/2 (Thr202/Tyr204) Assay Kit , PerkinElmer , ALSU-PERK-A500.

Techniques: Membrane, Knock-Out, Amplified Luminescent Proximity Homogenous Assay, Recombinant, Software

Journal: iScience

Article Title: A 2B adenosine receptor activation and modulation by protein kinase C

doi: 10.1016/j.isci.2023.107178

Figure Lengend Snippet: Probing the mechanisms of enhancement of cAMP accumulation by PMA treatment (A and B) Effect of PMA pretreatment on forskolin (A) and CTX (B)-induced cAMP accumulation in H9C2 cells. Cells were pretreated with PMA (1 μM) for 40 min followed by addition of forskolin (10 μM) and incubate for 20 min or CTX (0.5 μg/mL) for 60 min. PTX (200 ng/mL) was incubated with cells overnight. The bar graph is shown in mean ± S.E.M. of at least three individual experiments performed in triplicate. #p < 0.05, significantly different from CTX group (Student’s t test). CTX, cholera toxin. PTX, pertussis toxin. The mean value of maximal cAMP accumulation induced by 10 μM of forskolin was set as 100%. (C–F) Effects of kinase inhibitors and A 2B AR antagonists on enhancement of cAMP accumulation induced by PMA pretreatment. The maximum activation level of by NECA (10 μM) was set as 100%. The background of each individual cell type was set as 0%. Cells were incubated with PTX (200 ng/mL) overnight and with antagonists or inhibitors for 20 min before addition of PMA (1 μM) and incubated for 40 min, followed by addition of the A 2B agonist BAY and incubation continued for additional 20 min. PTX, pertussis toxin. The concentration of PSB603, MRS1754, GF109203X or GO6983 used was 1 μM; 10 μM H89 was used. (G) Time-course of PMA (1 μM)-induced increase of ERK1/2 activity. GO6983, H89, or PSB603 (1 μM) was added 20 min before PMA addition. Graphs shown are from three individual experiments performed in triplicate. ∗p < 0.05 (significantly different for the corresponding values in “+PMA” group, One-way ANOVA, Tukey’s post hoc test).

Article Snippet: AlphaScreen cAMP kit, SureFire®p-ERK1/2 (Thr202/Tyr204) Assay Kit , PerkinElmer , ALSU-PERK-A500.

Techniques: Incubation, Activation Assay, Concentration Assay, Activity Assay

Journal: iScience

Article Title: A 2B adenosine receptor activation and modulation by protein kinase C

doi: 10.1016/j.isci.2023.107178

Figure Lengend Snippet:

Article Snippet: AlphaScreen cAMP kit, SureFire®p-ERK1/2 (Thr202/Tyr204) Assay Kit , PerkinElmer , ALSU-PERK-A500.

Techniques: Membrane, Knock-Out, Amplified Luminescent Proximity Homogenous Assay, Recombinant, Software

Journal: Nature Communications

Article Title: Characterisation of IL-23 receptor antagonists and disease relevant mutants using fluorescent probes

doi: 10.1038/s41467-023-38541-2

Figure Lengend Snippet: a Left: Crystal structure of N -terminal domains 1–3 of IL23R (orange) in complex with IL-23 (IL23p19 = blue, IL12p40 = green) and the N -terminal domain of IL12Rβ1 (PDB: 6WDQ ). Right: The interface between IL23R and IL23p19 with C115 coloured cyan. b The phospho-STAT3 signal generated when cells expressing different constructs were treated with 5 nM IL-23. Data are mean ± SEM from five or six (untagged) or three (NanoLuc tagged) independent experiments conducted in triplicate. For the IL23R + IL12Rβ1 data set, one outlier (11108.7) was detected using the Grubbs test (with alpha = 0.001) and removed from the statistical analysis. Statistical significance of the C115Y mutation on the alphaLISA data was assessed using a 2-way ANOVA with Tukey’s multiple comparison test. *** p < 0.001. The p value for the comparison of NL tagged construct response was 0.0001 and the p value for the comparison of untagged receptor was 0.951. c Luminescence signal measured when IL12Rβ1 was expressed with either NL-IL23R or the C115Y mutant. Data are mean ± SEM generated from the mean luminescence values of seven (C115Y) or eight independent experiments performed in pentuplicate. d The proportion of luminescence from c that emanated from extracellular protein (as defined by the reduction in luminescence after treatment with 60 μM NanoLuc extracellular inhibitor). Data are mean ± SEM generated from the mean luminescence values of five (C115Y) or six independent experiments performed in triplicate. e The BRET ratio generated from HaloTag-618 labelled cells expressing NL tagged wildtype or C115Y IL23R with HT-IL12Rβ1 or unlabelled IL12Rβ1. Data are mean ± SEM generated from the mean BRET values of three independent experiments performed in pentuplicate. Statistical significance of differences in mean values shown in c – e were measured using a 2-sided, non-paired t test with ** indicating a p value of <0.005 and *** indicating a p value of <0.0005. The p values for the results shown in c , d and e were p = 0.148, 0.0005 and 0.0013 respectively. Source data are provided as a Source Data file.

Article Snippet: The following day media was replaced with serum free DMEM and the cells incubated for 3 h. Media was then replaced with HBSS containing 1 mg/ml BSA and increasing concentrations of IL-23 and the cells incubated for 30 min. An AlphaLISA SureFire Ultra assay kit (Perkin Elmer #ALSU-PST3) was then used to measure STAT3 phosphorylation at residue Tyr705.

Techniques: Generated, Expressing, Construct, Mutagenesis, Comparison