anti phospho camkii rabbit polyclonal igg antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: microRNA-544a as a new modulator of the Wnt-signalling network in the articular cartilage and osteoarthritis

doi: 10.1101/2024.11.28.625937

Figure Lengend Snippet: (a) Putative target genes of miR-544a associated with the activation of the Wnt pathway identified through TargetScan interrogation. b) Schematic representation of putative targets genes related to the β-catenin-dependent Wnt pathway. c) To validate some of these targets, 3’UTR-seed sequences of Axin-2, CDH1, CTNNBP1 and GSK3β or mutated sequences as control, were cloned in pmirGLO vectors upstream the Firefly luciferase gene. Confirming binding of miR-544a to these genes, reduced luminescence was recorded in C28/I2 cells transfected with miR-544a mimic (100 nM) and the vectors expressing the seed sequences 48h after transfection. No reduction in luminescence was reported in cells transfected with vectors expressing the mutated forms of the seed sequences. Results are expressed as mean ± SEM of Relative Luminescence units of Firefly luciferase normalized to Renilla luciferase (n=3). (d) mRNA expression levels of Axin-2, CDH1, CTNNBIP1 and GSK-3β in HACs transfected with miR-544a mimic (50nM) or negative control (NC, 50nM) for 3 hours, assessed by qPCR after 24 hours (n=6). qPCR results were normalised to the housekeeping gene β-actin and expressed relative gene expression (%) in comparison to NC. (d) Immunofluorescence analysis showing upregulation of β-catenin at protein level in HACs transfected with miR-544a mimic (50nM) in comparison to NC mimic (50nM). Fluorescence intensity of immunoreactive areas was quantified on the whole cell and nucleus after 24 hours upon transfection. (n=4). MiR-544a (50nM) enhanced the ability of Wnt3a (100ng/ml) to activate the SUPER8XTOPFlash reporter assay in (f) HEK293FT cells and (g) C28/I2 cells (n=3). (h) Immunofluorescence analysis showing upregulation of phospho-CamKII (pCamKII) expression in HACs response to transfection with miR-544a mimic in comparison to NC (both 50nM). Fluorescence intensity of immunoreactive areas was quantified on the whole cell and nucleus after 24 hours of transfection. (n=4). DAPI – positive staining for nuclei. Original magnification – x40, scale bar-50 µM. Data are shown as mean ± SEM. Statistical analysis was performed by one-way ANOVA or unpaired two-tailed t-test followed by Mann-Whitney post-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: After blocking, samples were incubated overnight at 4°C with the following antibodies: anti–phospho-CaMKII rabbit polyclonal IgG antibody (Cell Signalling Technology), anti-beta-catenin (Cell Signalling Technology), anti-MMP-13 mouse monoclonal antibody (Santa Cruz Biotechnology) or anti-Aggrecan Neoepitope polyclonal antibody (Invitrogen), all diluted 1:100 in blocking buffer.

Techniques: Activation Assay, Control, Clone Assay, Luciferase, Binding Assay, Transfection, Expressing, Negative Control, Comparison, Immunofluorescence, Fluorescence, Reporter Assay, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: microRNA-544a as a new modulator of the Wnt-signalling network in the articular cartilage and osteoarthritis

doi: 10.1101/2024.11.28.625937

Figure Lengend Snippet: (a, b) Immunofluorescence assay aimed to detect the expression of MMP-13 or MMP-cut aggrecan neoepitope in human articular chondrocytes (HACs) transfected with miR-544a mimic (50 nM) or NC (50 nM) in presence or absence of the β-catenin inhibitor Xav-939 (10 µM) or of the CaMKII inhibitor KN93 (10 µM) for 24 hours. Fluorescence intensity of immunoreactive areas was quantified on the whole cell (n=3). (c) Schematic representation of miR-544a modulation on the Wnt signalling pathway. DAPI – positive staining for nuclei. Original magnification – x40, scale bar-50 µM. Data are shown as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey post-test. ***p<0.001; ****p<0.0001; ns=non-significant.

Article Snippet: After blocking, samples were incubated overnight at 4°C with the following antibodies: anti–phospho-CaMKII rabbit polyclonal IgG antibody (Cell Signalling Technology), anti-beta-catenin (Cell Signalling Technology), anti-MMP-13 mouse monoclonal antibody (Santa Cruz Biotechnology) or anti-Aggrecan Neoepitope polyclonal antibody (Invitrogen), all diluted 1:100 in blocking buffer.

Techniques: Immunofluorescence, Expressing, Transfection, Fluorescence, Staining

Journal: iScience

Article Title: Lysophosphatidylcholine induced by fat transplantation regulates hyperalgesia by affecting the dysfunction of ACC perineuronal nets

doi: 10.1016/j.isci.2024.111274

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal Anti-CaMKII-α , Cell Signaling Technology , Cat# 3357S; RRID: AB_2070308.

Techniques: Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Software

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt , Mfn2 -null MEFs with vec , CaMKII-WT, or CaMKII-DN in the μ-slide. Time-lapse images were taken every 10 min for 17 hr and 50 min. MEFs were tracked for velocity quantification. Scale bar: 50 μm.

Article Snippet: Antibody , Anti-pan-CaMKII (rabbit polyclonal) , Cell Signaling Technology , #3362 , WB (1:1000).

Techniques: