Journal: The Journal of Biological Chemistry

Article Title: Biochemical analysis of a TrkB receptor mutation that causes a developmental epileptic encephalopathy

doi: 10.1016/j.jbc.2026.111152

Figure Lengend Snippet: Analysis of TrkB Y434C stably expressed in heterologous cells reveals abnormalities of protein expression and migration pattern on SDS-PAGE. Western blot analysis of TrkB protein in pooled and individual cell lines stably expressing WT and Y434C mutant. A , schematic of the TrkB protein domain structure, location of the Y434C mutation, and the protein regions recognized by the antibodies for TrkB. B , SDS-PAGE of pooled WT and Y434C cell lines reveals striking differences in migrating patterns. Cell lysates were prepared from the pooled stable cells expressing WT or Y434C mutant, and proteins were resolved on 8% SDS-PAGE. Western blots were probed with TrkB antibody recognizing the ectodomain of TrkB. Protein loading and transfer were monitored by probing the same blots with β-actin antibody. WT protein was robustly expressed and migrated mainly at approximately 145 kDa (mature form) and at 90 kDa (immature form) to a lesser extent. In contrast, the Y434C protein migrated mainly at approximately 90 kDa. C , quantification of 145 kDa ( left ) and 90 kDa ( right ) of TrkB protein levels from pooled WT or Y434C expressing cells from five independent experiments. The statistical analysis was performed by Student’s t test, unpaired two tailed. D , SDS-PAGE of representative subsets of individual WT and Y434C cell lines reveals striking heterogeneity of expression level and migrating pattern of mutant protein. Aliquots of lysates of selected individual cell lines were subjected to Western blot analyses using antibodies targeting either the N-terminal ectodomain ( top blot ) or the C-terminal (targeting the FLAG epitope on the C terminus) ( bottom blot ). The antibody targeting the N-terminal ectodomain revealed a prominent 145 kDa band in WT and in one Y434C mutant cell line (#6). In the remaining Y434C mutant cell lines, striking heterogeneity was evident in both expression levels and size (ranging from 40 to 100 kDa). With respect to the C-terminal, robust immunoreactivity was evident at 145 kDa in WT and one Y434C mutant stable cell line (#6). By contrast, there was no robust immunoreactivity in the remaining Y434C cell lines. E , twelve subclones from TrkB Y434C #24 stable cell line were further selected and analyzed by Western blot analysis using TrkB antibodies. The TrkB N-terminal antibody detected multiple immunoreactivities migrating at approximately 40 to 60 kDa ( top panel ). The level of immunoreactivity varied among 12 subclones. In contrast, C-terminal antibodies, including Trk and FLAG antibodies ( middle and bottom panels , respectively), failed to reveal a detectable level of immunoreactivity in these Y434C cell clones. Arrows in the right margin of the middle panel denote the presence of bands detected in nontransfected human embryonic kidney cells. β-actin blots in D and E were developed from separate gels. F , PCR detection of TrkB complementary DNA in Y434C mutant cells. Top , design of PCR strategy and primers amplifying TrkB DNA fragments corresponding to the intracellular domain of TrkB protein. Bottom , PCR amplification reveals the presence of TrkB DNA fragments from plasmid DNA ( left ) and genomic DNA isolated from Y434C #24 cell line. The dashed lines in D and F denote the positions at which irrelevant lanes were removed from the gel images.

Article Snippet: The following TrkB antibodies were employed in this study: N-terminal TrkB, mouse monoclonal (immunogen, amino acids 156–322), 1:500 dilution, catalog #610101, BD Transduction Laboratories; N-terminal TrkB, rabbit monoclonal (80E3) (immunogen, a synthetic short peptide surrounding amino acid 50), 1:1000 dilution, catalog #4603, Cell Signaling; C-terminal TrkB, rabbit monoclonal targeting FLAG epitope, 1:500 dilution, catalog #14793, Cell signaling; C-terminal TrkB, rabbit polyclonal (immunogen, a peptide corresponding to 14 amino acids between 783 and 796 of TrkB), 1:250 dilution, catalog #sc-11, Santa Cruz.

Techniques: Stable Transfection, Expressing, Migration, SDS Page, Western Blot, Mutagenesis, Two Tailed Test, FLAG-tag, Clone Assay, Amplification, Plasmid Preparation, Isolation