Generation and Validation of an Anti-Human PANK3 Mouse Monoclonal Antibody

Abstract

Coenzyme A (CoA) is an essential co-factor at the intersection of diverse metabolic pathways. Cellular CoA biosynthesis is regulated at the first committed step—phosphorylation of pantothenic acid—catalyzed by pantothenate kinases (PANK1,2,3 in humans, PANK3 being the most highly expressed). Despite the critical importance of CoA in metabolism, the differential roles of PANK isoforms remain poorly understood. Our investigations of PANK proteins as potential precision oncology collateral lethality targets (PANK1 is co-deleted as part of the PTEN locus in some highly aggressive cancers) were severely hindered by a dearth of commercial antibodies that can reliably detect endogenous PANK3 protein. While we successfully validated commercial antibodies for PANK1 and PANK2 using CRISPR knockout cell lines, we found no commercial antibody that could detect endogenous PANK3. We therefore set out to generate a mouse monoclonal antibody against human PANK3 protein. We demonstrate that a clone (Clone MDA-299-62A) can reliably detect endogenous PANK3 protein in cancer cell lines, with band-specificity confirmed by CRISPR PANK3 knockout and knockdown cell lines. Sub-cellular fractionation shows that PANK3 is overwhelmingly cytosolic and expressed broadly across cancer cell lines. PANK3 monoclonal antibody MDA-299-62A should prove a valuable tool for researchers investigating this understudied family of metabolic enzymes in health and disease.