The mRNA binding protein Fox3 (aka Rbfox3, Hrnbp3, A6nfn3) is now well known to be the same protein as NeuN, the "neuronal nuclear marker", first described by Mullen et al. in 1992. A mouse monclonal antibody, called A60, was cloned out because it had an interesting and potentially useful neuron specific staining pattern, though the protein target was unknown and appeared to be unrelated to the immunogen. The antibody was subsequently licensed to Millipore-Sigma who have marketed it for many years, see here. They noted that the A60 antibody bound the 46-48kDa bands on CNS homogenates but also a weaker band at about 70kDa. In 2009 Kim et al. identified NeuN protein as FOX3, one of the very large family of RNA binding proteins. By immunoprecipitation and mass spectroscopy they showed that A60 also binds synapsin 1, but with about 20 fold lower affinity compared to binding to FOX3. Despite this cross-reactivity they showed that A60 did not bind synaptic regions rich in synapsin 1 in stained sections. The epitope for A60 was mapped to within the N-terminal 106 amino acids of human FOX3 using recombinant constructs. These authors pointed out proline rich regions of sequence in synapsin 1 isotypes which were similar but not identical to sequences at the N-terminus of FOX3. Based on this similarity they proposed but did not prove that the A60 epitope was at the N-terminus of the molecule, the region of homology with synapsin 1. Later we mapped the epitopes for both A60 and our own mouse monoclonal FOX3 clone MCA-1B7 to the peptide YPPAQYPPPPQNGIPAEYAP, amino acids 5-24 of the human sequence, which is the region Kim et al. had postulated to contain the A60 epitope based solely on sequence homology and firmly mapping the A60 and MCA-1B7 epitope for the first time. Mao et al. 2016 studied the synapsin 1 crossreactivity of A60 in detail. Their figure 1 shows weak staining of synaptic regions in rat hippocampus with the original A60 antibody, but much less with the same antibody licensed from Zymed a company now part of ThermoFisher. This is of course a surprising result. They used VGLUT1 and synapsin I antibodies to label these same structures identifying them as mossy fibre terminals. Their figure 4 shows a western blot of A60 on hippocampus and cortical extracts revealing a band at about 70kDa along with the expected 46-48kDa doublet. They provide pretty compelling evidence that this band corresponds to synapsin 1, since the western blot image obtained with synapsin I antibody overlaps the A60 band perfectly. They also show that the A60 antibody, purchased from two different vendors reveals some puzzling problems. Also puzzling, on western blots synapsin 1 is expressed strongly in both hippocampus and cortex, while the very weak immunostaining of synapses was only seen in the hippocampus. They claim that a limited panel of other FOX3/NeuN antibodies do not show this curious hippocampal synaptic stain or cross reactivity with the 70kDa band on western blots.
The presence of another band at about 70kDa has been noted by many other workers, both using A60 and other antibodies raised against recombinant or peptide sequences of FOX3. Here is a rather awful blot from the very expensive and supposedly well respected Cell Signalling Technology here; This one, from Bioss has much stronger staining for the ~70kDa band than it does for the 46-48kDa doublet here; Here is another one from ThermoFisher showing something unexpected in heart tissues here; The iReal company has that band, but not in heart, see here; Western blot data from Hellobio is totally confusing here; Here's another antibody from German Research Products here;
We have worked up our MCA-1B7 to throw more light on this issue. We have already documented that our antibodies, MCA-1B7 and our various polyclonals, all bind only neuronal cells showing no binding to synapses. Clearly if any of them stained synapsin 1 efficiently synaptic regions would light up strongly which is obviously not the case. We showed that our antibody is more robust than A60, showing a higher titre and stronger kinetics of binding, and this data is on our web site. We also epitope mapped A60, MCA-1B7 and other available FOX3/NeuN monoclonals and found that the peptide YPPAQYPPPPQNGIPAEYAP, amino acids 5-24 of FOX3, strongly inhibits binding of both MCA-1B7, A60 and others to recombinant human FOX3. The mapping was done with 20 amino acid nested peptides overlapping by 5 amino acids and the data suggested that the central 10 amino acids of the peptide, YPPPPQNGIP, is likely the most significant component of the MCA-1B7 and A60 epitopes (see here for details). This is the same region which Kim et al. suggested was responsible for the cross-reactivity with synapsin I. Our in house studies showed that several other independently generated FOX3/NeuN monoclonals we made at the same time as MCA-1B7 also bound this region, suggesting that it is unusually immunogenic. So in conclusion current data suggests that the 70kDa band is synapsin 1 which shows up on blots since it is a very high abundance protein with low sequence homology to FOX3/NeuN. In contrast FOX3/NeuN is, like most RNA binding proteins, of very low abundance and is only revealed robustly sine A60, MCA-1B7 and others are high affinity reagents. The FOX3/NeuN epitope we defined is close to the N-terminus and so should be accessible to antibodies while the putative synapsin I epitope is buried within the sequence. This suggests that the sunapsin I epitope may be partially or wholly masked in fixed sections. In line with this suggestion, Mao et al. showed that saponin treatment of sections increased the A60 staining of hippocampal mossy fibres. My advice is don't worry about the 70kDa band as it has virtually no impact on cell and tissue staining. A60, MCA-1B7 and other FOX3/NeuN antibodies are very widely utilized as neuronal markers and have been since the Mullen et al paper came out in 1992.
