Fig. 6: Proximity-dependent biotin labeling (BioID) identifies a role for RASSF2 in regulation of Rac GTPase activation in AML.

a Schematic showing retroviral expression vectors and general assay principle for performing proximity-based biotin labeling and identification using an improved biotin ligase (BioID2, see Methods). b Strategy for performing proximity-based biotin labeling in Kasumi-1 t(8;21) cell line. Experiment was performed in three independent replicates that were submitted for mass-spectrometric analysis, and 60 unique high-confidence RASSF2-proximal proteins were identified across three replicates. c Bar graph showing significantly enriched Gene Ontology (GO) terms associated with the RASSF2-specific protein hits identified by proximity-based biotin labeling. d, Western blot showing interaction between HA-DOCK2 and Flag-RASSF2 as assessed in HEK293T cell lysates by immunoblotting following HA-immunoprecipitation (IP) ~48 h post-transfection with indicated constructs. Inputs are shown below. Molecular weight markers (kDa) are indicated at left. Data are representative of three experiments. e Immunoblotting for active (GTP-bound) Rac by PAK1 pull-down assay performed in two representative AML cell lines 4 days post-transduction with lentiviral shRNAs targeting control sequence (shCTRL) or RASSF2 (shR2#1, shR2#2) as indicated. Data are representative of three experiments. f Quantification of experiments performed in e. Data are normalized relative to β-actin protein abundance and presented as mean ± s.e.m. of three experiments.