Journal: The Journal of Biological Chemistry
Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures
Figure Lengend Snippet: Expression of wildtype (WT) proBDNF and its N121Q mutant (NQ) in stably transfected HEK293F cells. Protein expression was determined by Western blotting using an antibody, recognizing both BDNF and proBDNF, in the conditioned media (A) and cell lysates (B). Arrows point to (pro)BDNF forms: glycosylated proBDNF (black arrow), nonglycosylated proBDNF (white arrow), and mature BDNF (gray arrow). M indicates MagicMark molecular weight marker. Expression of mRNA was determined in WT and N121Q mutant by RT-PCR (C). ProBDNF/BDNF expression in cell lysates (D) or conditioned media (E) of HEK293F cells expressing N121Q mutant treated with proteasome inhibitor MG132 (MG), lysosomal inhibitor chloroquine (CQ), or chemical chaperone 4-phenylbutyric acid (PBA). The black arrow points to the position of nonglycosylated proBDNF.
Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).
Techniques: Expressing, Mutagenesis, Stable Transfection, Transfection, Western Blot, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction