Comparison of equivalence between two commercially available S499-phosphorylated FMRP antibodies in mice

Abstract

Fragile X syndrome (FXS) develops from excessive trinucleotide CGG repeats in the 5'-untranslated region at Xq27.3 of the Fmr-1 gene, which functionally silences its expression and prevents transcription of its protein. This disorder is the most prominent form of heritable intellectual deficiency, affecting roughly 1 in 5,000 males and 1 in 10,000 females globally. Antibody specificity and selectivity are essential for investigating changes in intracellular protein signaling and phosphorylation status of the Fragile X Mental Retardation Protein (FMRP). Currently, both PhosphoSolutions1 and abcam1produce commercially available S499-phosphorylated FMRP specific antibodies. The antibody from PhosphoSolutions1 has been validated in previous studies; however, the antibody from abcam1 antibody has yet to receive similar validation. This study aims to determine whether these two antibodies are true equivalents through western blot analysis of both NS-Pten knockout (KO) and Fmr-1 KO mice strains. We prepared hippocampal synaptosomal preparations and probed the samples using total FMRP, abcam1 phosphorylated FMRP, and Phospho-Solutions1 phosphorylated FMRP antibodies. We found that there was a significant increase in phosphorylated FMRP levels using the abcam1and PhosphoSolutions1 antibodies in the NS-Pten KO mice compared to wildtype mice. However, there was much more variability using the abcam1 antibody. Furthermore, there was a band present in the Fmr-1 KO for the phosphorylated FMRP site using the abcam1 antibody for western blotting but not for the PhosphoSolutions1 antibody. Our findings strongly suggest that the antibody from abcam1 is neither specific nor selective for its advertised targeted substrate, S499-phosphorylated FMRP.