The Hectd1 Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line offering a loss-of-function model for the E3 ubiquitin ligase Hectd1 in a mouse macrophage background. This product is provided as a ready-to-use cell line, enabling dissection of Hectd1??s role in ubiquitin-dependent protein degradation and signaling without transient knockdown or chemical inhibition.
RAW 264.7 cells are derived from BALB/c mouse macrophages transformed with the Abelson murine leukemia virus, yielding an adherent line that responds to LPS and IFN-??. These cells exhibit robust phagocytosis, cytokine production, and antigen presentation, making them a standard innate immunity model. Their stable growth and genetic tractability facilitate knockout-based functional studies.
Hectd1 encodes an E3 ubiquitin ligase that catalyzes ubiquitin transfer from E2 conjugating enzymes to substrate proteins, tagging them for 26S proteasomal degradation or altering signaling. Known substrates include Hsp90 and Dvl2, linking Hectd1 to Wnt/planar cell polarity and TGF-?? pathways. It is regulated by NF-??B downstream of LPS, TNF-??, and IFN-??, and modulates Smad7 stability, thereby influencing Smad4-dependent transcription and ??-catenin/TCF responses.
In macrophages, Hectd1 knockout likely disrupts ubiquitin-dependent regulation of inflammatory mediators and cytoskeletal dynamics. Loss of Hectd1 may impair degradation of Dvl2 and Hsp90, altering Wnt/PCP-directed migration and NF-??B signaling termination, potentially dysregulating TNF-?? and IL-6 secretion and phagocytosis. This cell line serves as a relevant system to examine E3 ligase dysfunction in inflammation, neural tube defects, and cancer.
The knockout cell line supports applications such as co-immunoprecipitation for substrate interactions, Western blotting of ubiquitinated proteins, NF-??B luciferase reporter assays, and cytokine ELISA. Functional assays include fluorescent bead phagocytosis, wound healing migration, and flow cytometry for surface markers during polarization. These enable investigation of E3 ligase function in immune cells, ubiquitin-dependent signaling in inflammation, and drug target validation. For further information, contact Ascent Research.
