Article Title: A Novel Peptide Interfering with proBDNF-Sortilin Interaction Alleviates Chronic Inflammatory Pain
Figure Lengend Snippet: Mapping of proBDNF-sortilin interaction interface. (A) Schematic diagram illustrating the design of human BDNF prodomain variants. Six variants with a series of amino acids deletions were used in this study. V66M, Val66Met; S, Signal peptide (amino acid 1-18); BDNF, mature BDNF (amino acid 129-247); HA, C-terminal HA epitope. (B) Immunoblots of co-immunoprecipitation (Left) or inputs (Right) of sortilin and proBDNF variants obtained with antibody against HA or Myc epitope. IP, Immunoprecipitation; IB, Immunoblot. (C) Left , live fluorescence micrograph of BiFC mapping assay (Yellow) using HEK293T cells with VC155-sortilin (Sort1) and VN155(I152L)-BDNF prodomain variants. Scale bar represents 50 µm. Right , quantification of fluorescence intensity of BiFC assay. P <0.0001, one-way ANOVA. ** P <0.01 versus Fl group; *** P <0.0001 versus Fl group; ## P <0.001 versus variant I; ### P <0.0001 versus variant I group; n.s., non-significant. Values are means ± SEM. n = 100 individual cells from 4 images. (D) Left , representative immunoblots of supernatant and cell lysates of HEK293 cells overexpressing sortilin and BDNF prodomain variants obtained with antibodies against HA. Right , densitometry analyses of secreted BDNF in supernatants, normalized to input in lysates. P <0.001, one-way ANOVA. * P <0.05, versus Fl group; *** P <0.001, versus Fl group; ## P <0.05, versus I group. Values are means ± SEM. n = 4. IB, Immunoblot. (E) BDNF ELISA quantification of supernatant collected from HEK293 cells overexpressing sortilin and BDNF prodomain variants. P <0.0001, one-way ANOVA. * P <0.05; ** P <0.01; *** P <0.001, versus Sort1/Fl group; ## P <0.05, versus Sort1/variant I group. Values are means ± SEM. Each data point represents the average of 3 independent experiments.
Article Snippet: The primary antibodies used included anti-proBDNF antibody (#ANT-006-AG, Alomone Labs Ltd, Jerusalem, Israel), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell signalling Technology, Danvers, MA, USA), anti-NeuN antibody (MAB377, EMD Millipore Corporation, Billerica, MA, USA) and anti-sortilin antibody (Abcam, Cambridge, UK).
Techniques: Western Blot, Immunoprecipitation, Fluorescence, Mapping Assay, Bimolecular Fluorescence Complementation Assay, Variant Assay, Enzyme-linked Immunosorbent Assay