A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture.

ABSTRACT:

Purpose

Primary open angle glaucoma-associated mutations in myocilin (MYOC) cause protein "nonsecretion," rendering secreted MYOC difficult to detect using conventional techniques. This study focused on developing and using an assay that can quickly and easily detect mutant MYOC secretion.

Methods

We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T and NTM-5 cells. Secreted and intracellular levels of MYOC eGLuc2 variants were evaluated by Western blotting and compared to untagged and FLAG-tagged MYOC constructs. Secreted and soluble intracellular MYOC eGLuc2 were measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from transfected cells to test the applicability of the assay for

SUBMITTER: Zadoo S 

PROVIDER: S-EPMC5110263 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature