Journal: iScience

Article Title: Genome-wide CRISPR-Cas9 screen analyzed by SLIDER identifies network of repressor complexes that regulate TRIM24

doi: 10.1016/j.isci.2023.107126

Figure Lengend Snippet:

Article Snippet: T7E1 assays were performed using the EnGen® Mutation Detection Kit per manufacturers protocol (NEB E3321S) and gene specific primers to amplify a ∼500bp region containing PAM site targeted by Synthego to perform knockouts.

Techniques: Recombinant, Mutagenesis, Protease Inhibitor, SYBR Green Assay, Plasmid Preparation, Cell Culture, Purification, TA Cloning, Bicinchoninic Acid Protein Assay, Software

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: Generation of RPE-PLK3 knock-out cells. ( A ) Schematic diagram of CRISPR/Cas9-mediated targeting of human PLK3 and of domain organization of PLK3 protein. Black arrowhead indicates the position of the critical ATP-binding Lysine 91 in the catalytic domain. Empty arrowhead indicates the position of the epitope of the PLK3 antibody from Cell Signaling. Fragments of the genomic DNA from selected clones were PCR amplified and analyzed by sequencing. ( B ) Parental RPE and selected clones of RPE-PLK3-KO cells were analyzed by immunoblotting using rabbit monoclonal PLK3 antibody (Cell Signalling). ( C ) Parental RPE and two independent clones of RP-PLK3-KO cells were probed with indicated antibodies. The position of PLK3 is indicated by an arrowhead. Please note the missing band corresponding to PLK3 in staining with antibody from St Johns laboratory and high level of cross-reactivity with other proteins.

Article Snippet: To generate PLK3 knock out cell line, RPE cells grown at 6-well plate were transfected with synthetic sgRNA (CRISPRevolution sgRNA EZ Kit; Synthego, Menlo Park, CA, USA) and recombinant EnGen Spy Cas9 NLS (New England Biolabs, Ipswich, MA, USA) using CRISPRMAX reagent (ThermoFisher Scientific).

Techniques: Knock-Out, CRISPR, Binding Assay, Clone Assay, Amplification, Sequencing, Western Blot, Staining