STUB1 Knockout Cell Line (Hela)

The choice depends on whether you are studying CHIP's role as a co-chaperone E3 ubiquitin ligase for HSP70/HSP90 client proteins or its functions in protein quality control. The Knockout line is the standard tool for asking whether CHIP is required for ubiquitination of misfolded HSP70/HSP90 clients — its principal characterized function in proteostasis. Overexpression is useful for testing whether elevated CHIP enhances client protein degradation or for studying CHIP mutations associated with autosomal recessive spinocerebellar ataxia 16 (SCAR16). For protein quality control research, the EDITGENE STUB1/CHIP Knockout in HeLa is a tractable platform with established HSP70/HSP90 client biology. Rescue with wild-type, U-box-deleted (E3 ligase-dead), or TPR-domain-mutant (chaperone-binding-deficient) CHIP enables dissection of E3 ligase versus co-chaperone functions. SCAR16-associated mutations can be introduced for disease modeling.

Primary applications: • HSP70/HSP90 client stability: cycloheximide chase analysis for HSP90 client proteins (ErbB2, AR, mutant p53, others) to assess CHIP-dependent degradation. • Protein quality control: misfolded protein clearance assays following thermal stress or proteasome inhibition challenges. • In vitro ubiquitination: reconstitution assays with purified CHIP and HSP70/HSP90-bound substrates for biochemical characterization. • SCAR16 disease modeling: rescue with autosomal recessive cerebellar ataxia-associated STUB1 mutations enables genotype-phenotype correlation studies. EDITGENE recommends this model for researchers investigating protein quality control biology, co-chaperone E3 ligase function, and SCAR16 disease mechanisms.

Yes. CHIP rescue experiments are well-established given the maturity of HSP70/HSP90 quality control research: • Construct design: use a codon-modified STUB1 sequence with a small C-terminal tag (FLAG, HA). CHIP's N-terminal TPR domain binds HSP70/HSP90 EEVD motifs and C-terminal U-box mediates E3 ligase activity — both must be preserved. • U-box-deleted rescue: U-box mutation or deletion abolishes E3 ligase activity while preserving co-chaperone binding — the standard control for distinguishing ligase from chaperone-modulating functions. • TPR-mutant rescue: TPR domain mutations (e.g., K30A) disrupt HSP70/HSP90 binding without affecting U-box activity, enabling dissection of substrate-targeting from catalytic functions. • SCAR16 disease mutation rescue: introduction of autosomal recessive cerebellar ataxia 16-associated STUB1 mutations enables genotype-function correlation studies. HeLa transduces efficiently with lentivirus and is one of the most widely used cell lines for protein quality control research, with extensive established protocols for CHIP rescue experiments.