Journal: Blood Advances

Article Title: A novel activating JAK1 mutation in chronic eosinophilic leukemia

doi: 10.1182/bloodadvances.2021004237

Figure Lengend Snippet: JAK1 pseudokinase mutations demonstrate transforming capability and result in hyperactivation of the JAK/STAT pathway. (A) Growth curves of Ba/F3 cells expressing JAK1 A634D and JAK1 R629_S632delinsSA cultured in the absence of IL-3 (graph is representative of 3 independent experiments). (B) Immunoblot analysis of Ba/F3 parental cells, Ba/F3 cells expressing JAK1 wild-type, JAK1 A634D, and JAK1 R629_S632delinsSA cultured in the presence of IL-3 and IL-3–independent Ba/F3 cells expressing JAK1 A634D, and JAK1 R629_S632delinsSA. (C) Immunoblot analysis of HEK293 cells expressing JAK1 wild type, JAK1 A634D, and JAK1 R629_S632delinsSA. p-, phosphorylated; WT, wild-type.

Article Snippet: Vectors, cloning, cell culture, retrovirus generation, Ba/F3 transformation assays, measurement of drug response by cell proliferation assay, and immunoblotting JAK1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002227.2","term_id":"102469033","term_text":"NM_002227.2"}} NM_002227.2 ) variant c.1887_1894delinsTG, p.Arg629_Ser632delinsSerAla, and c.1901C>A, p.A634D pDONR vectors were generated in pDONR223-JAK1 vector (Addgene plasmid #23932).

Techniques: Expressing, Cell Culture, Western Blot

Journal: Blood Advances

Article Title: A novel activating JAK1 mutation in chronic eosinophilic leukemia

doi: 10.1182/bloodadvances.2021004237

Figure Lengend Snippet: JAK1 pseudokinase mutations demonstrate sensitivity to JAK inhibitors. (A) Representative graphs of the dose-response curves (72-hour sensitivity) for different JAK inhibitors on Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA (upper panels). Graphs showing mean IC50 (lower left panel) and area under the dose-response curve (AUC; lower right panel) for different JAK inhibitors on Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA (3 independent experiments). (B) Immunoblot analysis of IL-3–independent Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA treated with vehicle or 50 nM or 100 nM of ruxolitinib for 4 hours (n = 2 replicates). (C) Sensitivity of peripheral blood and bone marrow specimens from the patient with JAK1 R629_S632delinsSA to JAK inhibitors, represented by IC50, in comparison with control samples obtained from healthy donors (HD) and specimens from a larger cohort of patients with MPNs) (the horizontal lines denote median IC50 of HD and MPN specimens). ****P < .0001, ***P < .001, 1-way analysis of variance. p-, phosphorylated.

Article Snippet: Vectors, cloning, cell culture, retrovirus generation, Ba/F3 transformation assays, measurement of drug response by cell proliferation assay, and immunoblotting JAK1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002227.2","term_id":"102469033","term_text":"NM_002227.2"}} NM_002227.2 ) variant c.1887_1894delinsTG, p.Arg629_Ser632delinsSerAla, and c.1901C>A, p.A634D pDONR vectors were generated in pDONR223-JAK1 vector (Addgene plasmid #23932).

Techniques: Expressing, Western Blot, Comparison, Control

Journal: bioRxiv

Article Title: Systematic part transfer by extending a modular toolkit to diverse bacteria

doi: 10.1101/2023.02.07.527528

Figure Lengend Snippet: A . Design of the new destination vectors containing KanR, the pBBR1 origin and rep gene, and a CIDAR cloning site where transcription units may be assembled. Although this is over 100 parts, only the parts in this study are shown. B . Molecules of Equivalent Fluorescein (MEFL) expression values for the combinations of parts included in the original CIDAR paper. These were interpolated from Supplementary Figure 1. Expression from the original vector series in E . coli matches closely to the expression reported in this study. C . Violin plots of the expression values in MEFL for E. coli populations expressing each construct, along with the average copy number assessed by ddPCR. The violin plots for three replicates are overlaid, showing little variation between replicates. D . The same plots in as in C for the same constructs expressed in P. putida . E . The same plots in as in C for the same constructs expressed in C. necator . F . The same plots in as in C for the same constructs expressed in K. nataicola .

Article Snippet: The CIDAR MoClo Parts Kit (#1000000059) and CIDAR MoClo Extension, Volume I (#1000000161) were purchased from Addgene.

Techniques: Cloning, Expressing, Plasmid Preparation, Construct