anti-cacnb2 antibody  (Alomone Labs)

Journal: International Journal of Surgery (London, England)

Article Title: Comprehensive analysis of an ATF/CREB family-based signature with regard to prognosis and immune feature in adrenocortical carcinoma: a cohort study

doi: 10.1097/JS9.0000000000002961

Figure Lengend Snippet: Flow diagram of patient inclusion, signature construction, and validation in ACC cohorts. A schematic overview of the study design including sample screening, inclusion, and analytical stages. Adrenocortical carcinoma (ACC) patients from three independent cohorts – The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and West China Hospital (WCH) – were assessed. After applying exclusion criteria, final cohorts included TCGA ( n = 79), GEO ( n = 24), and WCH ( n = 78). A transcription factor-based prognostic signature involving ATF/CREB family genes was constructed using Cox and LASSO Cox regression in the TCGA training cohort. The model’s prognostic and immunological significance was analyzed. External validation was performed in GEO ( GSE10927 ) and WCH cohorts, with further functional assays in SW13 cell line.

Article Snippet: The human ACC cell line SW13 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Biomarker Discovery, Gene Expression, Construct, Functional Assay

Journal: Nature chemical biology

Article Title: Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

doi: 10.1038/s41589-023-01530-z

Figure Lengend Snippet: Fig. 3 | ASO–protein interactions studied by Drug-ID. a, Drug-ID was applied to an RNaseH-recruiting gapmer. For covalent conjugation to SNAP-eGFP- BASU, a benzylguanine moiety was attached. b, Schematic overview of uptake/ conjugation and SILAC–MS/MS experiments. c, PAGE analysis of BG–ASO uptake into transgenic HeLa cells. Conjugation of the BG–ASO was visualized by conjugation of a competing fluorescent BG–FITC probe. Western blot served as loading control (n = 1). d, SILAC–MS/MS for 10 nM BG–ASO against 10 nM control ASO. Plotted was the enrichment (log2) of replicate 1 against replicate 2 with swapped SILAC labeling. Statistical significance of hits was calculated with respect to the distance of the median of the distribution of all protein ratios as well as protein intensities using Perseus (significance threshold P < 0.05). e, Fluorescence microscopy of ASO and biotin ligase. BG–ASO was transfected into transgenic HeLa cells expressing SNAP-eGFP-BASU (green channel). For visualization, 2 nM Atto594-labeled ASO (red channel) was spiked

Article Snippet: For this, 2 × 105 HeLa cells (DSMZ, no: ACC 57) were seeded in DMEM/FBS in a 12-well plate.

Techniques: Conjugation Assay, Multiplex sample analysis, Tandem Mass Spectroscopy, Transgenic Assay, Western Blot, Control, Labeling, Fluorescence, Microscopy, Transfection, Expressing

Journal: Nature chemical biology

Article Title: Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

doi: 10.1038/s41589-023-01530-z

Figure Lengend Snippet: Fig. 4 | isASO-ID overcomes intracellular localization limitations of Drug-ID. a, Microscopy as in Fig. 3e. LNA-modified and MOE-modified ASO (red channel) were transfected at a final concentration of 30 nM (10 nM BG–ASO and 20 nM control ASO) into transgenic HeLa cells expressing SNAP-eGFP-BASU (green channel). White arrows indicate co-localization of Atto594–ASO and the biotin ligase (n = 1). b, SILAC–MS/MS for 10 nM BG–ASO and 20 nM control ASO for the LNA-modified ASO against Lipofectamine 3000. Plotted was the enrichment (log2) of replicate 1 against replicate 2 with swapped SILAC labeling (n = 2). c, SILAC–MS/MS for 10 nM BG–ASO and 20 nM control ASO for the LNA-modified ASO over the MOE-modified ASO. Plotted was the enrichment (log2) of replicate 1 against replicate 2 with swapped SILAC labeling. Statistical significance of hits in b and c was calculated with respect to the distance of the median of the distribution of all protein ratios as well as protein intensities using Perseus. Significance was set to P < 0.05 in b and c. d, Schematic overview of isASO-ID experiments. e, Fluorescence microscopy using Halo-BASU-His for biotinylation. Either 25 nM MOE-modified or LNA-modified Halo-ASO (20-mer)

Article Snippet: For this, 2 × 105 HeLa cells (DSMZ, no: ACC 57) were seeded in DMEM/FBS in a 12-well plate.

Techniques: Microscopy, Modification, Transfection, Concentration Assay, Control, Transgenic Assay, Expressing, Multiplex sample analysis, Tandem Mass Spectroscopy, Labeling, Fluorescence

Journal: Nature chemical biology

Article Title: Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

doi: 10.1038/s41589-023-01530-z

Figure Lengend Snippet: Fig. 5 | isASO-ID identifies the ASO interactome more comprehensively. a, Determining localization of ASO and biotinylated proteins via fluorescence microscopy. Increasing amounts of BG–ASO (15, 25, 50 and 100 nM; 20-mer MOE) were transfected (with Lipofectamine 3000) into wild-type HeLa cells. Microscopy was carried out as for Fig. 4e (n = 2). b, Exemplary competition experiment with the ASO drug. Overlaid was the enrichment plot for 15 nM BG–ASO against 25 nM control ASO in two replicates (blue), with the enrichment plot for the competition experiment 15 nM BG–ASO + 85 nM control ASO against 100 nM control ASO in two replicates (orange). c, Immunofluorescence microscopy for 15 nM BG–ASO (20-mer MOE) and the influence of ActD treatment (1.5 µg ml−1 for 1 h) on ASO localization and its co-localization with NONO. On the right, the influence of ActD treatment on biotin deposition is shown under the same conditions (n = 2). d, SILAC–MS/MS experiment for 15 nM BG–ASO (20-mer MOE) treated with ActD plotted against 15 nM control ASO

Article Snippet: For this, 2 × 105 HeLa cells (DSMZ, no: ACC 57) were seeded in DMEM/FBS in a 12-well plate.

Techniques: Fluorescence, Microscopy, Transfection, Control, Immunofluorescence, Multiplex sample analysis, Tandem Mass Spectroscopy