Journal: bioRxiv

Article Title: The human AMPKγ3 R225W mutation does neither enhance basal AMPKγ3-associated activity nor glycogen in human or mouse skeletal muscle

doi: 10.1101/2023.08.28.555048

Figure Lengend Snippet: To understand the function of the AMPKγ3 R225W protein, m. tibialis anterior (TA) of a single lower hindlimb was contracted in wild-type (WT, white bar) and homozygote AMPKγ3 R225W carrier (KI HOM, blue bar) mice while the contralateral lower hindlimb served as a sham-operated resting control. AMPKα2βγ1 ( A-B ) and AMPKα1βγ1 ( C-D ) activity was measured in the sham-operated or contracted state without or with 200 µM AMP added to the activity assay (n=8-11). Glycogen levels in the sham and contracted muscles ( E , n=8-10). Protein expression of TBC1D1 ( F ), ACC ( G ), p-P38 Thr180/Tyr182/P38 ( H ), p38 ( I ), pERK 1/2 Thr202/Tyr204/ERK ( J ) and ERK ( K ) was measured in sham or contracted TA (n=8-9). The amount of AMPKα2 ( L ) and AMPKγ3 ( M ) in the AMPKγ3 immunoprecipitates (n=8-10). Data are given as means + SEM including univariate scatterplots to indicate individual values. All Western blotting data are normalized to WT Sham levels. A two-way ANOVA (A-M) with Student-Newman-Keuls post-hoc test was used to evaluate differences between genotype, contraction or the interaction. *: Indicates effect of contraction compared to sham within genotype. #: Indicates difference between genotypes within contraction-state. #: p<0.05, ***/###: p<0.001. A.U.: Arbitrary Units. ME: Main Effect.

Article Snippet: Primary antibodies against pACC Ser212/222 (3661, mouse/human), pAMPK Thr172 (2531), Erk1/2 (9102), pErk1/2 Thr202/Tyr204 (9101), p38 (9212), p-p38 Thr180/Tyr182 (9211), Akt2 (3063), pAkt Thr308 (9275) and pTBC1D4 Thr649 (8881) were from Cell Signaling Technology (Danvars, MA, USA).

Techniques: Activity Assay, Muscles, Expressing, Western Blot

Journal: Cancers

Article Title: The Impact of ETV6-NTRK3 Oncogenic Gene Fusions on Molecular and Signaling Pathway Alterations

doi: 10.3390/cancers15174246

Figure Lengend Snippet: Activation of signaling pathways by EN variants and their potential mechanisms in oncogenic transformation. ( A ) Activation of the ERK pathway, assessed using a transcription factor ELK1-responsive luciferase reporter. ( B ) Western blot quantification of ERK1/2 (T202/Y204) phosphorylation levels, 24 h post-induction in generated stable cell line lysates, revealing the intensity of ERK phosphorylation across different EN variants. ( C ) Activation of the STAT1 signaling pathway determined using a luciferase assay with a STAT1-responsive element. ( D ) Activation of the STAT3 signaling pathway assessed using a luciferase reporter assay with a STAT3-responsive element, indicating the potential role of EN variants in STAT3-mediated cellular processes. ( E ) Assessment of the JNK pathway activation by EN variants, using an AP-1-responsive luciferase reporter assay. ( F ) Mass cytometry-derived mean values of phosphorylation activation sites of ribosomal protein S6 (S235/S236), STAT1 (Y701), STAT3 (Y705) and STAT5 (Y694) detected at the single-cell level in stable cell lines, normalized to a control. The data show the relative intensity of signaling events initiated by each EN variant. The EN2 cell line without tetracycline induction was employed as the reference control for the normalization of stable cell line experiments. Luciferase assay column graphs individual measurements are shown as black dots and the level of statistical significance of the EN variant values compared to NTRK3 values, is denoted by asterisk-symbols: * ( p < 0.05), ** ( p < 0.005), *** ( p < 0.0005), **** ( p < 0.00005) or “ns” for not significant ( p > 0.05). See for original Western Blots.

Article Snippet: Phosphorylated ERK1/2 was detected using a primary pERK1/2 (T202/Y204) antibody (Cell signaling technology #9101, 1:1000 dilution in 5% BSA—in 0.05% Tween).

Techniques: Activation Assay, Transformation Assay, Luciferase, Western Blot, Generated, Stable Transfection, Reporter Assay, Mass Cytometry, Derivative Assay, Variant Assay