Other NLG sites were difficult to identify due to the presence of multiple NLG sites and the lack of proteolytic cleavage sites within the peptide GSK2118436 sequence region . Therefore, we focused on the Asn900 and Asn913 residues to evaluate site-specific NLG differences between the figitumumab-sensitive and resistant cells. A complete peptide fragmentation patterns of the tryptic peptide contained formerly N-glycosylated peptide at the Asn900 was observed from both sensitive and resistance cells, which encompassed the Asn residue of the glycosylation site at Asn900 . These results demonstrated that Asn900 was glycosylated in both drug-sensitive and resistant cells. However, Enzalutamide side effects peptides with NLG at Asn913 were identified only in the sensitive, but not resistant cells, suggesting that this specific NLG site was not glycosylated in the resistant cells. To further verify the NLG consensus site and its functional importance, a site-directed IGF1R mutant was constructed. Asn913 was replaced with a glutamine residue to yield an N913Q mutant. To assess the functional consequences of this mutation, wild-type IGF1R and the mutant construct were transiently expressed in Huh7 cells. As shown in Figure 5B, the expression levels of wild-type and mutant IGF1R in the transfected cells were increased remarkably compared to cells transfected with the empty pcDNA3.1 vector. However, the N913Q mutation appeared as a 105 kDa band that migrated faster than the wild-type protein which produced a similar migration pattern of the protein on SDS-polyacrylamide gel electrophoresis in SNU719 cells. These observations confirmed that the,115 kDa band in sensitive cells corresponded to IGF1R that was NLG at N913. Interestingly, it seems that removal of N-linked sugars from N913 of the IGF1R had no apparent effect on the formations of IGF1R/IR HRs. Rather, this mutation only affected on the NLG state of the receptor because HR levels were remarkably increased in the mutant IGF1R-transfected cells and the mutant receptor showed an increased migration rate on SDS-PAGE . This result suggested that removal of the N-linked sugar from the N913 site altered the SDS-PAGE banding profile of IGF1Rb but had no effect on the heterodimerization of IGF1R and IR. We next performed an immunofluorescence assay to determine whether mutation of the N913 consensus site prevented cell surface expression of IGF1R. Cells expressing wild-type IGF1R had an abundance of plasma membrane-bound IGF1R whereas the mutant form was primarily retained inside the cells with relatively little or no plasma membrane localization . To assess the functionality of NLG-deficient-IGF1Rs compared to the wild-type form, we performed MTT assays. The results showed that the anti-proliferative effect of figitumumab was increased by overexpressing wild-type IGF1R, whereas cells transfected with the mutant IGF1R did not display any changes in drug sensitivity .