Antibody crossreactivity between the tumour suppressor PHLPP1 and the proto‐oncogene β‐catenin

Abstract

During recent years, the PH domain leucine‐rich repeat protein phosphatase 1 (PHLPP1) has received increasing attention as a tumour suppressor that functions by dephosphorylating and antagonizing the survival‐promoting protein kinases AKT, PKC and S6K1 [1]. Previous studies reported that PHLPP1 co‐localizes with the tumour suppressor Scribble at cell–cell contacts [2], its expression is lost in colorectal cancer tumours [3] and it correlates with expression of PTEN, a tumour suppressor phosphatase that functions as a negative regulator upstream from AKT [2,4]. We warn that the PHLPP1 antibodies used in these studies (Bethyl IHC‐00382 and A300‐660A) crossreact strongly with β‐catenin, a proto‐oncogene product known to localize to adherens junctions. When probing Caco‐2 whole cell lysates (WCLs) for PHLPP1 with commonly used commercial PHLPP1 antibodies (Bethyl IHC‐00382 and A300‐660A—different types of the same antibody), we observed an additional prominent immunoreactive band around 90 kDa (Fig 1C), which was still apparent on PHLPP1 immunoprecipitation using the same antibodies (Fig 1A). In contrast with full length PHLPP1, the 90 kDa band was not responsive to siRNA‐mediated knockdown of PHLPP1 (Fig 1A,C), arguing against a PHLPP1 variant and suggesting it reflected antibody crossreactivity. To characterize further this putative crossreactivity, we used stable isotope labelling in cell culture and …