customized phospho-specific antibody microarray  (Full Moon BioSystems)

Journal: JCI Insight

Article Title: Targeting PI3K γ anchoring enhances CFTR membrane localization and modulator efficacy via PKD1

doi: 10.1172/jci.insight.198846

Figure Lengend Snippet: ( A ) Experimental workflow used for phosphoproteomic analysis of F508del-CFBE41o- cells treated with control peptide (CP) or PI3Kγ MP (25 μM, 30 minutes). A phospho-specific microarray, containing 875 phosphosite-specific and 451 pan-specific antibodies, was used. Thirty-six proteins showing phosphorylation changes exceeding ±60% compared with the control (expressed as percentage CFC, i.e., percentage fold-change compared with control) were selected for downstream analysis. ( B and C ) Panther Gene Ontology (GO) slim-term enrichment analysis of proteins with altered phosphorylation after PI3Kγ MP treatment. Significantly enriched GO terms (FDR < 0.05) were categorized under ( B ) biological processes and ( C ) cellular components. ( D ) Reactome pathway enrichment analysis of differentially phosphorylated proteins after PI3Kγ MP treatment. Lines represent the top 20 pathways; x axis shows the –log 10 (FDR). Color intensity reflects fold enrichment, and circle size indicates the number of proteins; color intensity (yellow to red) indicates increasing fold enrichment. The full phospho-array protein list served as background reference. ( E ) Proteins with CFC greater than 60% (green) or less than −60% (red). A CFC of 100% corresponds to a 2-fold increase in signal intensity after PI3Kγ MP treatment relative to CP.

Article Snippet: After treatment with 25 μM PI3Kγ MP or an equimolar amount of CP for 30 minutes, F508del-CFBE41o- cells were lysed as described above, and protein samples were frozen at –80°C before being subjected to an antibody microarray (KAM-1325 array) and data analysis, which was performed at Kinexus.

Techniques: Control, Microarray, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Multi-omics analysis reveals that ALYREF-mediated m 5 C modification promotes platinum resistance in ovarian cancer via the NSUN2/ALYREF/LGR4 axis

doi: 10.1038/s41419-025-08310-8

Figure Lengend Snippet: A Schematic diagram of the ALYREF m 5 C methylation binding site mutation. B m 5 C-RIP and qRT-PCR assays detecting the influence of ALYREF on the m 5 C methylation level of LGR4 mRNA. C qPCR detection of LGR4 RNA expression in the influence of ALYREF. D Western blot analysis of LGR4 protein expression in ALYREF-overexpressing A2780/DDP and SKOV3/DDP cells, and in cells with mutated ALYREF binding sites. E Actinomycin D assay assessing the impact of ALYREF on LGR4 mRNA stability. F Representative images of IHC staining of LGR4 expression levels in tissue microarrays. G Correlation analysis between ALYREF and LGR4 protein expression levels. H Schematic representation of the subcutaneous tumor model in cisplatin-resistant ovarian cancer-bearing mice by Generic Diagramming Platform (GDP) . I Final diameter and weight of the subcutaneous tumors. Data are displayed as mean ± SD; *: p < 0.05. **: p < 0.01. ***: p < 0.001. ****: p < 0.0001.

Article Snippet: Following a rinse with PBS, the tissue microarrays were incubated at 4 °C for 16 h with anti-ALYREF antibody (16690-1-AP; Proteintech, China) and anti-LGR4 antibody (20150-1-AP; Proteintech, China), each at a 1:300 dilution.

Techniques: Methylation, Binding Assay, Mutagenesis, Quantitative RT-PCR, RNA Expression, Western Blot, Expressing, Immunohistochemistry