Fig. 5
Western and Lectin Blot of O-glycosylated Sec-G-CSF. a Western blot detection of Sec-G-CSF:cMyc (Secretory variant, 28 kDa). Samples collected at 4 and 6 dpi. Four leaf discs from three different plants were collected for each sample. 20 µg TSP of PBS samples were loaded on the gel. Black arrow denotes monomeric Sec-G-CSF:cMyc. G-CSF: protein standard. Proteins were detected with G-CSF antibody. b Band quantification of Western blot detected Sec-G-CSF:cMyc. c c-Myc Detection: 0.25 µL eluent (Sec-G-CSF without glyco-machinery: -) after c-Myc purification; and 2.5 µL eluent (10x more than negative control) (Sec-G-CSF with glyco-machinery: +) after c-Myc purification. Detection using the c-Myc antibody. GalNAc Detection (Lectin Blot):: Equalized amounts of c-Myc purified Sec-G-CSF (without and with glyco-machinery: -, +). GalNAc detection using VVA-HRP. GelCode Blue: GelCode Blue Staining of c-Myc purified Sec-G-CSF (without and with glyco-machinery: -, +). Sec-G-CSF:cMyc co-expressed with p19 and glyco-machinery (∓) (O-Glycosylation machinery: UDP-GlcNAc/GalNAc epimerase, UDP-GlcNAc/GalNAc transporter, and GalNAc-glycosyltransferase) (OD600 ratios; Sec-G-CSF:cMyc: 0.5; p19: 0.1; GNT2: 0.2; epimerase/transporter: 0.1/strain). Columns denoted with a different letter are significantly different (p ≤ 0.05) using one-way ANOVA and followed by Tukey test. Error bars are standard deviation of the means