Journal: Oncogenesis

Article Title: A functional CRISPR/Cas9 screen identifies kinases that modulate FGFR inhibitor response in gastric cancer

doi: 10.1038/s41389-019-0145-z

Figure Lengend Snippet: a Western blot confirmed that ILK and CSK gene expression was effectively knocked down in KatoIII cells. Cells were transfected with 10 nmol/l oligos and were harvested 72 h. ILK (#3856) and CSK (#4980) antibodies were obtained from Cell Signaling. Vinculin (#13901, Cell Signaling) was the loading control. b Upon AZD4547 treatment, knocking down ILK decreased cell viability while knocking down CSK increased cell viability. c Knocking down ILK or CSK alone does not affect KatoIII cell viability. KatoIII cells were seeded in 6-well plates at 300,000 cells/well the day before transfection and were transfected with 10 nM siRNA oligos using LipoJet (SignaGen Laboratories). The Dicer-substrate short interfering RNAs (DsiRNAs) targeting CSK (CSK_1 Reference #146599035 and CSK_2 #146599032), ILK (ILK_1 Reference #146599024 and ILK_2 #146599021) as well negative control oligos (NTC) were obtained from IDT. The viability was measured 6 days by CellTiter-Glo after treated with AZD4547 or plain median. Representative data from one of three independent experiments are shown. The data are measured in hextuple as mean ± s.e.m. **** P < 0.0001; ** P < 0.01. The P values were calculated by Dunnett’s multiple comparisons test

Article Snippet: Cells were transfected with 10 nmol/l oligos and were harvested 72 h. ILK (#3856) and CSK (#4980) antibodies were obtained from Cell Signaling.

Techniques: Western Blot, Gene Expression, Transfection, Control, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic lupus erythematosus immune complexes increase the expression of SLAM family members CD319 (CRACC) and CD229 (LY-9) on plasmacytoid dendritic cells and CD319 on CD56(dim) NK cells.

doi: 10.4049/jimmunol.1301022

Figure Lengend Snippet: FIGURE 5. pDCs lack the activating adaptor molecules but express inhibitory adaptor molecules of SLAM receptors. (A) IFN-a production by RNA-IC–stimulated pDCs after cross-linking of CD319, CD229, or BDCA-2 by plate-bound mAbs. IFN-a levels were determined in 20-h cell culture supernatants by an immunoassay and normalized to the IFN-a production in cultures without plate-bound mAb. The dashed line indicates 100%. Data (mean + SD) are from three donors from two independent experiments. Expression of EAT2 and SAP (B) and SHIP-1, SHP-1, SHP- 2, and CSK (C) in cell lysates from isolated pDCs or NK cells from two healthy donors was determined by Western blot. Expression of b-actin was used as control.

Article Snippet: Rabbit polyclonal Abs to Ewing’s sarcoma-activated transcript 2 (EAT2), SHIP-1, SHP-2, CSK (Proteintech Group, Chicago, IL), and a rabbit mAb to SHP-1 (EPR5519; Epitomics, Burlingame, CA), followed by HRP-conjugated goat anti-rabbit IgG (H+L) (Invitrogen), were used to detect the indicated signaling molecules.

Techniques: Cell Culture, Expressing, Isolation, Western Blot, Control

Journal: Biochemistry

Article Title: An open library of human kinase domain constructs for automated bacterial expression

doi: 10.1021/acs.biochem.7b01081

Figure Lengend Snippet: Kinase domain constructs with yields >2 μ g/mL culture for 96-kinase expression screen. Kinases are listed by Uniprot designation and whether they were co-expressed with Lambda or truncated YopH164 phosphatase. Yield (determined by Caliper GX II quantitation of the expected size band) reported in μ g/mL culture, where total eluate volume was 120 μ L from 900 μ L bacterial culture. Yields are shaded green (yield > 12 μ g/mL), yellow (12 > yield > 7 μ g/mL) and orange (yield <7 μ g/mL); kinase domain constructs with yields that were undetectable or < 2 μ g/mL are not listed. ‡ denotes that the second kinase domain of KS6A1_HUMAN was expressed; all other kinases were the first or only kinase domain occurring in the ORF. Construct boundaries are listed in UniProt residue numbering for the UniProt canonical isoform. An interactive table of expression yields and corresponding constructs is available at http://choderalab.org/kinome-expression

Article Snippet: CSK_HUMAN , 186–450 , Addgene 23941 , YopH , 62.5.

Techniques: Construct, Expressing, Quantitation Assay, Residue, Plasmid Preparation

Journal: Frontiers in Pharmacology

Article Title: Decoding monocyte heterogeneity in sepsis: a single-cell apoptotic signature for immune stratification and guiding precision therapy

doi: 10.3389/fphar.2025.1675887

Figure Lengend Snippet: Development and validation of the nomogram-based predictive model (A) Nomogram diagnostic prediction model constructed using G0S2, ITM2A, PAG1, and GZMA. (B) Calibration curves assessing consistency between predicted and actual observed values. (C) Decision Curve Analysis (DCA) evaluating the clinical net benefit of the nomogram. The x-axis represents the threshold probability for diagnosing sepsis, while the y-axis represents the net benefit. The curve for our model (red line) shows a greater net benefit across a wide range of clinically relevant thresholds compared to the strategies of treating all patients (dashed gray line) or no patients (solid blue line), indicating its potential for clinical utility. (D) Clinical impact curve illustrating the practical application of the nomogram. At any given risk threshold on the x-axis, the red curve shows the number of individuals predicted to be high-risk, while the blue curve shows the number of true positive cases within that group. The close approximation of the two curves demonstrates the model’s strong performance in accurately identifying patients at high risk of sepsis in a clinical setting. (E–H) ROC curve analyses evaluating predictive accuracy of the model in the training and multiple validation datasets.

Article Snippet: The PVDF membranes were incubated overnight at 4 °C with primary antibodies targeting G0S2 (Abcam, ab236113; 1:1000), GZMA (Proteintech, 11288-1-AP; 1:1000), ITM2A (Proteintech, 14407-1-AP; 1:1000), and PAG1 (Proteintech, 25029-1-AP; 1:1000).

Techniques: Biomarker Discovery, Diagnostic Assay, Construct

Journal: Cell reports

Article Title: GDF6-CD99 Signaling Regulates Src and Ewing Sarcoma Growth

doi: 10.1016/j.celrep.2020.108332

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used: rabbit anti-FLI-1 (ab15289, Abcam); rabbit anti-GDF6 (Novus, NBP 1-91934); rabbit anti-caspase 3 (9665, Cell Signaling Technology); rabbit anti-PARP (sc-7150, Santa Cruz Biotechnology); mouse anti-CD99 (MS-1633, Lab Vision); sheep anti-CD99L2 (AF5185, R&D Systems); mouse anti-p21 (BD PharMingen, 556430); mouse anti-tubulin (Developmental Studies Hybridoma Bank); rabbit anti-GAPDH (sc-25778, Santa Cruz Biotechnology); rabbit anti-CSK (HPA028425, Atlas Antibodies); mouse anti-GST (MS-707-P0, Lab Vision); mouse anti-GFP (sc-9996, Santa Cruz Biotechnology); rabbit anti-actin (8457, Cell Signaling Technology); rabbit anti-Src (2123, Cell Signaling Technology); rabbit anti-phospho-Src (2101, Cell Signaling Technology); rabbit anti-phospho-Cortactin (4569, Cell Signaling Technology); rabbit anti-phospho-p130 Cas (4011, Cell Signaling Technology); rabbit anti-phospho-STAT3 (9145, Cell Signaling Technology); rabbit anti-phospho-Smad1/5 (9516, Cell Signaling Technology); rabbit anti-HA (3724, Cell Signaling Technology); and mouse anti-FLAG (F1804, Sigma-Aldrich).

Techniques: Control, Virus, Recombinant, Transfection, Reverse Transcription, SYBR Green Assay, In Situ, Labeling, Plasmid Preparation