Comparative Analysis of MBNL1 Antibodies: Characterization of Recognition Sites and Detection of RNA Foci Colocalization

<b>Background/Objectives:</b> MBNL1 is an RNA-binding protein involved in RNA metabolism, including splicing. It colocalizes with RNA foci, a pathological hallmark of myotonic dystrophy, and plays a central role in its disease mechanism. Moreover, MBNL1 has been implicated in other neuromuscular disorders and cancers. In these pathological and biochemical studies, the detection of MBNL1 using antibodies is essential. Given that MBNL1 has multiple splicing-derived isoforms, different antibodies may recognize distinct isoforms. This study aims to compare six commercially available antibodies regarding their specificity in Western blotting, colocalization with RNA foci, and suitability for immunoprecipitation. <b>Methods</b>: Western blot analysis was performed using MBNL1 isoforms and deletion mutants expressed in HEK293 cells, as well as endogenous MBNL1 from various cell lines. RNA fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were conducted in DM1 model cells and patient-derived fibroblasts to assess MBNL1 colocalization with RNA foci. Immunoprecipitation experiments were performed in HEK293 cells to evaluate antibody suitability for protein isolation. <b>Results</b>: Western blot analysis revealed that different antibodies target distinct regions of MBNL1, with three recognizing exon 3 and the remaining antibodies recognizing exon 4, exon 5, and exon 6, respectively. In the FISH-IF experiments, the clarity of RNA foci colocalization varied depending on the antibody used, with some antibodies failing to detect colocalization. The immunoprecipitation analysis showed that four antibodies were able to isolate endogenous MBNL1. <b>Conclusions</b>: This study clarifies the recognition properties and application suitability of MBNL1 antibodies, providing a valuable resource for research on MBNL1-related diseases and RNA metabolism.