Production and characterization of a highly specific and potent antibody capable of detecting endogenous RhebL1 protein in mammalian tissues and cell lines

Rheb (Ras homolog enriched in brain) is a small G-protein and a critical component of the insulin/TSC/mTOR pathway involved in the regulation of cell growth. Although a single gene for Rheb exists in most species, in mammals, two genes have been identified- Rheb1 and RhebL1 (Rheb2). Most studies to date have explored the expression and localization patterns of Rheb protein in mammalian cell lines using exogenously expressed tagged versions of Rheb1 for lack of a suitable antibody capable of detecting endogenous Rheb. The commercial antibodies available are either not able to detect the endogenous protein or are not specific enough to distinguish between the two Rheb proteins. We have developed and characterized a highly specific and potent affinity-purified antibody toward the mouse RhebL1 protein. This antibody does not cross react with Rheb1 and is highly sensitive as it can detect endogenous RhebL1 protein. Using this antibody, we have shown that RhebL1 protein is not ubiquitously expressed in mouse tissues and that the brain exhibits the highest level of expression. In addition, RhebL1 is expressed in two cell lines-3T3-L1 and L1C2, and its expression is induced in 3T3-L1 cells as they undergo differentiation from pre-adipocytes to adipocytes. This antibody now serves as a useful tool to study RhebL1 protein exclusively without any non-specific cross-reactivity with the Rheb1 protein.