Figure 2

ASP-2 post-translational modifications; glycosylation and disulphide linkages Panel A: ASP-2 samples were denatured by boiling in the presence of SDS and prior to treatment with PNGase, according to the manufacturer's protocol. Glycosylated and deglycosylated samples are depicted. A reduction in size of ~15-k is seen in the presence of PNGase due to the removal of N-linked sugars. Note the presence of higher ASP-2 bands, indicated with double arrows, which are similarly, deglycosylated. Panel B: ASP-2 samples were denatured in the presence or absence of a mixture of 10 mM DTT and 2% (vol/vol) β-mercaptoethanol designated as reducing agent. A reduction in the amount of the largest ASP-2 protein (marked with 3 arrows) is noted in the presence of reducing agent as well as a reduction in the mobility of ASP-2 proteins. Ni²⁺-affinity purified ASP-2 was used at a concentration of 40 nM. Western blots were detected with anti-ASP-2. Bold arrows designate larger putative, dimer and multimer.