Box 1 Western blot detection of ADNP

Over the years, ADNP detection has remained far from unambiguous and different antibodies against the protein have been raised. Initially, ADNP was discovered as a novel Activity-Dependent Neurotrophic Factor (ADNF9/14)-like protein with a neuroprotective capacity exceeding that of ADNF9 itself. Here, ADNP was visualized on Western blot after incubation with an antibody raised against ADNF-14 (SALLRSIPA) [9, 60]. Based on its amino acid sequence, the theoretical molecular weight of ADNP without posttranslational modifications is estimated at 124 kDa (https://www.uniprot.org/). However, this SALLRSIPA antibody detecting ADNF-14 together with the NAP sequence resulted in a specific band signal of only 90 kDa at a Western blot. A molecular weight of around 90 kDa as observed on Western blot thus suggests proteolytic processing of ADNP. In 2001, a specific antibody raised against the synthetic peptide based on the ADNP sequence 989 to 1015 (CEMKPGTWSDESSQSEDARSSKPAAKK) resulted in a single band signal at 114 kDa, still well below the predicted molecular mass of ADNP [14]. Later studies reported visualization of ADNP Western blots with variable molecular weights ranging from 114 to 150 kDa, stressing the urgent need for more standardized and reproducible ADNP detection methods [13, 12, 30, 33, 41, 61, 62]