Fig 1: Human WN1316-activating factors exert WN1316-mediated cytoprotective activity.Differentiated SH-SY5Y cells were treated with 8 µM WN1316 in the presence of purified GST-fusion proteins for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 µM menadione for 4 h. Concentrations of the GST-fusion proteins used are as follows; GST; 4.4 µg/ml, GST-RBP4; 2.2 µg/ml, GST-AHSG; 1.9 µg/ml, GST- SERPINA1; 2.4 µg/ml, GST-ITIH4; 2.3 µg/ml, GST-A1BG; 1.5 µg/ml, GST-HPX; 1.7 µg/ml, GST-CFB; 3.3 µg/ml. As control experiments, differentiated SH-SY5Y cells were treated with 8 µM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA (p<0.0001) followed by Dunnett’s post hoc test compared with GST-RBP4 (**p<0.001).
Fig 2: Silver staining and Western blot analysis of Blue pass fraction treated with or without glycosidase.Blue pass fraction was treated with or without EnzMix (removal N- and O-glycans) and PNGaseF (removal of N-glycans) at 37°C for 16 h. The molecular masses and homogeneities of the proteins were analyzed by SDS-PAGE (A) and Western blotting with anti-AHSG antibody as a glycoprotein standard (B). Proteins were visualized by silver staining using a Silver Stain Kit Wako (Wako).
Supplier Page from Abcam for Anti-AHSG antibody