Comparison of Equivalence between Two Commercially Available S499-Phosphorylated FMRP Antibodies in Mice

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 PhosphoSolutions(®) and abcam® produce commercially available S499-phosphorylated FMRP specific antibodies. The antibody from PhosphoSolutions(®) has been validated in previous studies; however, the antibody from abcam(®) 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, abcam(®) phosphorylated FMRP, and PhosphoSolutions(®) phosphorylated FMRP antibodies. We found that there was a significant increase in phosphorylated FMRP levels using the abcam® and PhosphoSolutions(®) antibodies in the NS-Pten KO mice compared to wildtype mice. However, there was much more variability using the abcam(®) antibody. Furthermore, there was a band present in the Fmr-1 KO for the phosphorylated FMRP site using the abcam(®) antibody for western blotting but not for the PhosphoSolutions(®) antibody. Our findings strongly suggest that the antibody from abcam(®) is neither specific nor selective for its advertised targeted substrate, S499-phosphorylated FMRP.