“… Figure 5 Functional validation of the knock-in clones (A) Western blot of HA tag, KANSL3 and GAPDH in three Kansl3 degron knock-in clones and wild type ES cells with and without dTAG-13 treatment for 24 h. An unspecific band is indicated with an asterisk. (B) Western blot of HA-tagged KANSL3 and GAPDH in Kansl3 degron knock-in cell line at different times after dTAG-13 addition ( Radzisheuskaya et al., 2021 ). (C) Bar plots representing mean ± SD fold cell expansion and percent of Trypan blue positive cells after 4 days of dTAG-13 treatment in Kansl3 degron knock-in cell line ( Radzisheuskaya et al., 2021 ).…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“… (B) Western blot of HA-tagged KANSL3 and GAPDH in Kansl3 degron knock-in cell line at different times after dTAG-13 addition ( Radzisheuskaya et al., 2021 ). (C) Bar plots representing mean ± SD fold cell expansion and percent of Trypan blue positive cells after 4 days of dTAG-13 treatment in Kansl3 degron knock-in cell line ( Radzisheuskaya et al., 2021 ). (D) Western blot of HA tag and GAPDH in dTAG-TAK1 expressing glioma stem cells treated with 500 nM dTAG13-NEsG, dTAG-13 or dTAG V -1.…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“… (B) Western blot of HA-tagged KANSL3 and GAPDH in Kansl3 degron knock-in cell line at different times after dTAG-13 addition ( Radzisheuskaya et al., 2021 ). …”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…We use knockin of an endogenous Kansl3 degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types. For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021) .…”
mentioning
confidence: 99%
“…For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021) .…”
Summary
Protein degradation technologies represent a powerful functional genomics tool, allowing fast and controllable target protein depletion. Establishing these systems requires a knock-in of the degradation tag into both endogenous target gene alleles. Here, we provide a step-by-step protocol for the efficient generation of biallelic degradation tag knock-ins in mouse and human cell lines using CRISPR-Cas9. We use knockin of an endogenous
Kansl3
degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types.
For complete details on the use and execution of this protocol, please refer to
Radzisheuskaya et al. (2021)
.
“… Figure 5 Functional validation of the knock-in clones (A) Western blot of HA tag, KANSL3 and GAPDH in three Kansl3 degron knock-in clones and wild type ES cells with and without dTAG-13 treatment for 24 h. An unspecific band is indicated with an asterisk. (B) Western blot of HA-tagged KANSL3 and GAPDH in Kansl3 degron knock-in cell line at different times after dTAG-13 addition ( Radzisheuskaya et al., 2021 ). (C) Bar plots representing mean ± SD fold cell expansion and percent of Trypan blue positive cells after 4 days of dTAG-13 treatment in Kansl3 degron knock-in cell line ( Radzisheuskaya et al., 2021 ).…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“… (B) Western blot of HA-tagged KANSL3 and GAPDH in Kansl3 degron knock-in cell line at different times after dTAG-13 addition ( Radzisheuskaya et al., 2021 ). (C) Bar plots representing mean ± SD fold cell expansion and percent of Trypan blue positive cells after 4 days of dTAG-13 treatment in Kansl3 degron knock-in cell line ( Radzisheuskaya et al., 2021 ). (D) Western blot of HA tag and GAPDH in dTAG-TAK1 expressing glioma stem cells treated with 500 nM dTAG13-NEsG, dTAG-13 or dTAG V -1.…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“… (B) Western blot of HA-tagged KANSL3 and GAPDH in Kansl3 degron knock-in cell line at different times after dTAG-13 addition ( Radzisheuskaya et al., 2021 ). …”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…We use knockin of an endogenous Kansl3 degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types. For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021) .…”
mentioning
confidence: 99%
“…For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021) .…”
Summary
Protein degradation technologies represent a powerful functional genomics tool, allowing fast and controllable target protein depletion. Establishing these systems requires a knock-in of the degradation tag into both endogenous target gene alleles. Here, we provide a step-by-step protocol for the efficient generation of biallelic degradation tag knock-ins in mouse and human cell lines using CRISPR-Cas9. We use knockin of an endogenous
Kansl3
degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types.
For complete details on the use and execution of this protocol, please refer to
Radzisheuskaya et al. (2021)
.
Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain‐containing protein 4 (YEATS4) is an essential gene for BC cell viability using CRISPR‐Cas9 library screening is reported, and that HUWE1 is an E3 ligase responsible for YEATS4 ubiquitination and proteasomal degradation by the Protein Stability Regulators Screening Assay. KAT8‐mediated acetylation of YEATS4 impaired its interaction with HUWE1 and consequently prevented its ubiquitination and degradation. The protein levels of YEATS4 and KAT8 are positively correlated and high levels of these two proteins are associated with poor overall survival in BC patients. Importantly, suppression of YEATS4 acetylation with the KAT8 inhibitor MG149 decreased YEATS4 acetylation, reduced cell viability, and sensitized BC cells to cisplatin treatment. The findings reveal a critical role of the KAT8/YEATS4 axis in both tumor growth and cisplatin sensitivity in BC cells, potentially generating a novel therapeutic strategy for BC patients.
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