Journal: Communications Biology
Article Title: Quassinoid analogs with enhanced efficacy for treatment of hematologic malignancies target the PI3Kγ isoform
Figure Lengend Snippet: Brusatol can directly target the PI3Kγ isoform. a IC50 of Brusatol was determined in these NPC cell lines (C17, NPC43, and NPC53) and Brusatol-sensitive SU-DHL-4 cells at 72 h post-treatment. b These cell lines were harvested and detected the endogenous expressions of indicated proteins with western blot analysis. c The lysates of SU-DHL-4 cells were incubated with biotin-conjugated 51048 compound together in the absence or presence of Brusatol, then the immunoprecipitated complex was detected with western blot. d In vitro expressed and purified GST-tagged PI3Kγ protein was incubated with 0, 300 μM, or 500 μM biotin-conjugated 51052 compound as well as Dynabeads M-280 Streptavidin. Then the binding protein was examined with western blot analysis. e A schematic diagram shows the target site of PIK3CG genes using the CRISPR/Cas9 system. f A Surveyor mutation detection assay was performed to verify whether the PIK3CG gene was mutated in knock-out (KO) Raji cells. The control cell line (sgVec) by transfecting empty plasmids were used as control. The yellow arrows indicated the truncated fragments. g The indicated proteins were detected in knock-out Raji cells by western blot analysis. h Knock-out Raji cells were untreated or treated with 100 nM of Brusatol for 72 h, then cells were harvested and determined the expressions of PI3K/AKT associated proteins with western blot.
Article Snippet: Pull-down assayThirty microgram of purified GST-tagged proteins were pre-cleared with Dynabeads M-280 Streptavidin (Invitrogen, Carlsbad, CA, USA).
Techniques: Western Blot, Incubation, Immunoprecipitation, In Vitro, Purification, Binding Assay, CRISPR, Mutagenesis, Detection Assay, Knock-Out