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: ACS Synthetic Biology

Article Title: A User’s Guide to Golden Gate Cloning Methods and Standards

doi: 10.1021/acssynbio.2c00355

Figure Lengend Snippet: Complete List of Golden Gate Toolkits Following the Common Syntax a

Article Snippet: MoClo CRISPR/Cas Toolkit for Plants , Addgene toolkit 1000000159 , ( ) , CRISPR/Cas nucleases and gRNA backbones. , SpeR or KanR , BsaI.

Techniques: Marker, Transformation Assay, Expressing, Selection, Plasmid Preparation, Infection, Clone Assay, CRISPR

Journal: ACS Synthetic Biology

Article Title: A User’s Guide to Golden Gate Cloning Methods and Standards

doi: 10.1021/acssynbio.2c00355

Figure Lengend Snippet: Complete List of Golden Gate Toolkits Following the Common Syntax a

Article Snippet: Mobius Assembly Vector Toolkit , Addgene toolkit 1000000134 , ( ) , An alternative set of Level 0 backbones using AarI as assembly enzyme, and chromogenic dropouts ( amilCP , spisPink , and sfGFP ) instead of lacZα to remove the need for X-Gal and IPTG. Also, an alternative method for hierarchical assembly based on 4-part cycling (as opposed to the 2-part cycling used in GoldenBraid). , CamR , BsaI.

Techniques: Marker, Transformation Assay, Expressing, Selection, Plasmid Preparation, Infection, Clone Assay, CRISPR