Fig. 5: LacNAc-dependent interaction of Ctg and HdgΔN with Drpr.
a, b Schematic diagrams showing full-length Ctg (a), and full-length Hdg and N-terminus-truncated HdgΔN (b). c, d S2 cells were transfected with drpr-HA and GFP-ctg or GFP-Lec24Db (c), or with drpr-HA and FLAG-hdg or FLAG-GFP (d). Cell lysates immunoprecipitated (IP) with HA antibodies were subjected to Western blot (IB) analyzes by GFP, HA or FLAG antibodies. e, f S2 cells were transfected with GFP-ctg and drpr-HA or drprNQ4-HA (e) or with FLAG-hdgΔN and drpr-HA or drprNQ4-HA (f). Cell lysates were subjected to immunoprecipitation by HA antibodies and to Western blot analyzes using HA, GFP and FLAG antibodies. Bar graphs showing quantification of GFP-Ctg, e or FLAG-HdgΔN, f level normalized to respective Drpr-HA or DrprNQ4-HA level, with averages (mean ± SEM) shown from Western blots. Statistical significance was determined by Student’s t test, two tailed with ** representing p < 0.01. p = 0.0076 in (e), and p = 0.00227 in (f). g, h Western blots showing transfected S2 cell lysates immunoprecipitated by HA antibodies for Drpr-HA in the presence of 0, 10, 100 and 1000 μM of 3’-O-sialic acid-LacNAc. The HA immunoprecipitates were immunoblotted with anti-GFP for GFP-Ctg (g) or anti-FLAG for FLAG-HdgΔN (h). c–h the experiments were repeated at least 3 times independently with similar results.
