Journal: Oncogenesis

Article Title: PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma

doi: 10.1038/s41389-024-00511-8

Figure Lengend Snippet: A Accumulation of MAPK, PI3K/AKT, YAP and PKC signalling effectors including total and phosphorylated DUSP6, MARCKS, ERK, YAP, S6 and AKT 24 h after treatment with vehicle control (−) or 10 nM Trametinib (+). Western blot analyses were performed using three independent biological replicates ( n = 3). kD kilodalton. REVERT total protein loading stain is shown in Supplementary Fig. . B Expression levels of pS6 and DUSP6 (normalised to total S6 or REVERT staining, respectively) in vehicle control or 10 nM Trametinib-treated GNAQ/GNA11 -mutant (solid dot) or wild-type (crossed dot) UM cell lines. Data derived from three independent biological experiments ( n = 3), and P values were calculated using paired t tests. ns not significant. C Percentage of cells undergoing S-phase inhibition (dotted line set at 50% S-phase inhibition) and change in % sub-G1 (dotted line set at 30% sub-G1) in GNAQ/GNA11 -mutant (solid circle) or wild-type (crossed circle) UM cell lines treated with 5 µM IDE196 (data derived from ), 10 nM Trametinib or 2 µM BEZ235. Data derived from three independent biological experiments ( n = 3).

Article Snippet: Membranes were incubated at 4 °C overnight in primary antibodies diluted in Intercept Blocking Buffer (TBS) (Li-Cor, Lincoln, NE, USA) or Odyssey Blocking Buffer (Li-Cor) with Tween 20 (0.05%), as follows: total MARCKS (1:1000, 2C2, WH0004082M6, Sigma-Aldrich), phosphorylated MARCKS (pMARCKS; Ser 152/156 , 1:1000, 2741 S, Cell Signaling Technology, Danvers, MA, USA), DUSP6 (1:250 or 1:1000, EPR129Y, ab76310, Abcam, Cambridge, UK), total AKT (1:1000, 40D4, 2920S, Cell Signaling Technology), phosphorylated AKT (pAKT; Ser 473 , 1:100, D9E, 4060S, Cell Signaling Technology), pAKT (Ser 473 , 1:500, 736E11, 3787, Cell Signaling Technology), total ribosomal S6 (1:500, 54D2, 2317 S, Cell Signaling Technology), phosphorylated ribosomal S6 (pS6; Ser 235/236 , 1:1000, 2F9, 4856S, Cell Signaling Technology), total YAP (1:500, 1A12, 12395S, Cell Signaling Technology), phosphorylated YAP (pYAP; Ser 127 , 1:2000, 4911, Cell Signaling Technology), total ERK (1:2 000, 137F5, 4695S, Cell Signaling Technology), phosphorylated ERK (pERK; Tyr 204 , 1:250, E-4, SC-7383, Santa Cruz, Dallas, TX, USA), and MEK1/2 (1:500, L38C12, 4694S, Cell Signaling Technology).

Techniques: Control, Western Blot, Staining, Expressing, Mutagenesis, Derivative Assay, Inhibition

Journal: Oncogenesis

Article Title: PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma

doi: 10.1038/s41389-024-00511-8

Figure Lengend Snippet: A Fold change in DUSP6 and pS6 expression (normalised log 2 protein expression in drug-treated – normalised log 2 protein expression in control-treated cells) in GNAQ/GNA11 -mutant (solid circle) and wild-type (crossed circle) UM cell lines treated with 5 µM IDE196, 10 nM Trametinib, or 2 µM BEZ235. Data compared using one-way ANOVA with the Geisser–Greenhouse correction and Tukey’s multiple comparison test, adjusted P values are shown. Data derived from three independent biological experiments ( n = 3, mean ± SD). B Accumulation of MAPK, PI3K, YAP and PKC signalling effectors, including total and phosphorylated DUSP6, MARCKS, ERK, YAP, S6 and AKT 24 h after treatment with vehicle control (−) or 2 µM BEZ235 (+). Western blot analyses were performed using three independent biological replicates ( n = 3). kD kilodalton. REVERT total protein loading stain is shown in Supplementary Fig. .

Article Snippet: Membranes were incubated at 4 °C overnight in primary antibodies diluted in Intercept Blocking Buffer (TBS) (Li-Cor, Lincoln, NE, USA) or Odyssey Blocking Buffer (Li-Cor) with Tween 20 (0.05%), as follows: total MARCKS (1:1000, 2C2, WH0004082M6, Sigma-Aldrich), phosphorylated MARCKS (pMARCKS; Ser 152/156 , 1:1000, 2741 S, Cell Signaling Technology, Danvers, MA, USA), DUSP6 (1:250 or 1:1000, EPR129Y, ab76310, Abcam, Cambridge, UK), total AKT (1:1000, 40D4, 2920S, Cell Signaling Technology), phosphorylated AKT (pAKT; Ser 473 , 1:100, D9E, 4060S, Cell Signaling Technology), pAKT (Ser 473 , 1:500, 736E11, 3787, Cell Signaling Technology), total ribosomal S6 (1:500, 54D2, 2317 S, Cell Signaling Technology), phosphorylated ribosomal S6 (pS6; Ser 235/236 , 1:1000, 2F9, 4856S, Cell Signaling Technology), total YAP (1:500, 1A12, 12395S, Cell Signaling Technology), phosphorylated YAP (pYAP; Ser 127 , 1:2000, 4911, Cell Signaling Technology), total ERK (1:2 000, 137F5, 4695S, Cell Signaling Technology), phosphorylated ERK (pERK; Tyr 204 , 1:250, E-4, SC-7383, Santa Cruz, Dallas, TX, USA), and MEK1/2 (1:500, L38C12, 4694S, Cell Signaling Technology).

Techniques: Expressing, Control, Mutagenesis, Comparison, Derivative Assay, Western Blot, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Nuclear Localization of α1A‐Adrenergic Receptors Is Required for Signaling in Cardiac Myocytes: An “Inside‐Out” α1‐AR Signaling Pathway

doi: 10.1161/JAHA.113.000145

Figure Lengend Snippet: α1‐ARs activate PKC in nuclei isolated from adult cardiac myocytes. A, Nuclei were isolated from adult cardiac myocytes and stained with antibodies to the PKCα, δ, or ε isoforms followed by DAPI counterstain (scale bar=5 μm). B, PKCα, δ, or ε isoform distribution in membrane (M), cytosolic (C), and nuclear (N) was measured by Western blot analyses. Fraction purity was verified by Western blots for NCX (membrane), GAPDH (cytosolic), and LAP2 (nuclear). Relative distribution of the PKC isozymes is presented as mean±SEM for 3 separate nuclear preparations. C, PKC activation was measured in isolated nuclei treated with PE (20 μmol/L, 2 μmol/L of timolol) by phosphorylation of PKCδ at Thr505. PKC activity was measured by phosphorylation of the PKC substrate, MARCKS (pMARCKS). LAP2, a nuclear marker, was used as a loading control. Quantitation of PKCδ phosphorylation at Thr505 and MARCKS phosphorylation is presented as mean±SEM from 3 separate nuclear preparations. Data were analyzed by Student's t test, and significant comparisons are indicated as P <0.05. GAPDH indicates glyceraldehyde 3‐phosphate dehydrogenase; LAP2, lamin‐associated protein 2; NCX, Na + /Ca 2+ exchanger; PE, phenylephrine; PKC, protein kinase C; pMARCKS, phospho‐myristoylated alanine‐rich C kinase substrate; α1‐ARs, α1‐adrenergic receptors.

Article Snippet: To determine nuclear PKC activity by Western blot, an antibody against phospho‐myristoylated alanine‐rich C kinase substrate (pMARCKS) was used (1:1000; Cell Signaling).

Techniques: Isolation, Staining, Membrane, Western Blot, Activation Assay, Phospho-proteomics, Activity Assay, Marker, Control, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Isosaponarin Derived from Wasabi Leaves on Glutamate Release in Rat Synaptosomes and Its Underlying Mechanism

doi: 10.3390/ijms23158752

Figure Lengend Snippet: Isosaponarin decreases 4-AP-evoked phosphorylation of SNAP-25 and MARCKS. Upper panel, Western blot bands of SNAP-25, pSNAP-25 (Ser187), pMARCKS (Ser152/156), and β-actin. Lower panel, expressions of SNAP-25, pSNAP-25 (Ser187), and pMARCKS were normalized with β-actin. Isosaponarin was added 10 min before the addition of 4-AP. Data are presented as mean ± SEM (n = 5 per group). *** p < 0.001 vs. control group; # p < 0.001 vs. 4-AP-treated group.

Article Snippet: The primary antibodies used were PKC (1:600, Abcam, Cambridge, UK); pPKC (1:1000; Cell Signaling, Beverly, MA, USA); PKCα (1:600; Cell Signaling, Beverly, MA, USA); pPKCα (1:2000; Abcam, Cambridge, UK); SNAP-25 (1:20,000; Abcam, Cambridge, UK); pSNAP-25 (Ser187) (1:2000; Abcam, Cambridge, UK); pMARCKS (Ser152/156) (1:250; Cell Signaling, Beverly, MA, USA); and β-actin (1:1000; Cell Signaling, Beverly, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Control