PPP1R16B Knockout HAP1 Cell Line

The choice depends on whether you are studying PPP1R16B (TIMAP, TGF-β inhibited membrane-associated protein)'s role as a membrane-associated PP1 regulatory subunit or its functions in endothelial barrier function and pulmonary vascular biology. The Knockout line is appropriate for asking whether TIMAP is required for PP1 targeting to membrane-associated substrates — TIMAP contains ankyrin repeats and is membrane-anchored via N-terminal myristoylation. Overexpression is useful for studying TIMAP in endothelial contexts. For endothelial biology research, the EDITGENE PPP1R16B Knockout in HAP1 is a clean genetic background, though physiological TIMAP function in endothelial barrier requires endothelial cell models. TIMAP has been characterized in pulmonary endothelial cell barrier function and is downregulated by TGF-β. Rescue with wild-type or myristoylation-deficient (G2A) TIMAP enables membrane targeting studies.

Primary applications: • PP1 holoenzyme assembly: co-immunoprecipitation analysis of PP1 catalytic subunits with TIMAP to characterize membrane-localized holoenzyme. • Membrane targeting studies: imaging analysis of myristoylation-dependent membrane localization in rescue cell lines. • Endothelial barrier biology: in endothelial-relevant contexts (heterologous), studies of TIMAP-PP1's role in endothelial junctions and TGF-β-regulated barrier function. • Substrate identification: phosphoproteomics to identify candidate TIMAP-targeted PP1 substrates. EDITGENE recommends this model for researchers investigating membrane-targeted PP1 biology and TIMAP-specific substrate identification.

Yes. TIMAP rescue experiments require attention to membrane targeting: • Construct design: use a codon-modified PPP1R16B sequence with a small C-terminal tag (FLAG, HA). TIMAP has N-terminal myristoylation site (G2) and ankyrin repeats — preserve N-terminus for membrane targeting. • Myristoylation-deficient rescue: G2A mutation abolishes membrane targeting and generates cytoplasmic TIMAP for membrane-localization studies. • PP1-binding-deficient rescue: RVxF motif mutations enable PP1-dependent versus -independent function dissection. • Functional readout: rescue should restore plasma membrane localization, PP1-TIMAP holoenzyme formation, and membrane-localized substrate dephosphorylation. HAP1-specific considerations: • Diploidization: HAP1 cells gradually diploidize during extended culture — confirm ploidy by flow cytometry at the time of phenotypic assay. • Integration site sensitivity: position effects on transgene expression are more pronounced in near-haploid backgrounds; generating multiple independent rescue clones is strongly recommended. • Transduction efficiency: HAP1 transduces with lentivirus at moderate efficiency — increase MOI compared to standard immortalized lines.