Additionally, there was no detectable insulin-dependent heterodimer formation or dissociation due to figitumumab. Phosphorylation in response to 100 nMinsulin was also not reduced by figitumumab . Taken together, the results from this experiment demonstrated that IGF1R/IR heterodimers responded well to IGF1, and blocking of IGF1 by figitumumab induced the down-regulation of IGF1 Dabrafenib Raf inhibitor ligand-dependent IGF1R/IR heterodimer formation in the drug-sensitive cell lines. To evaluate whether the effect of figitumumab was restricted to SNU719, HepG2, or SNU368 cells, we performed studies in cells with low expression levels of IR, including SNU739 and SNU886 cells, transfected with pcDNA3.1-IR which induced high expression of IR. As shown in Figure 3A, IR expression levels in the transfected cells increased remarkably compared to cells transfected with the pcDNA3.1 empty vector. Moreover, IGF1R/IR HRs were also formed in the IR-transfected cells. To examine whether IGF1R/IR HR formation due to increased IR protein levels could enhance drug sensitivity, we performed MTT assays. The result showed that IR-transfected cells were more sensitive to the increased anti-proliferative effect of figitumumab . These results indicated that elevated levels of IR and IGF1R enabled cancer cells to form IGF1R/IR HRs and increased their anti-proliferative response to figitumumab. Aside from the association between HRs and drug sensitivity, we also found that N-linked glycosylation is an additional important factor that influences the response to figitumumab. Blotting for the anti-IGF1Rb subunit revealed two isoforms around 95 and 105 kDa in most cells; however, sensitive cells showed a weak 105 kDa band and a stronger bands at 115 kDa . In short, IGF1Rb in figitumumab-sensitive cells migrated more slowly on SDS-PAGE than that in resistant cells. Interestingly, blotting for the anti-IRb subunit produced the same band pattern as that of IGF1Rb. In order to determine whether differences in molecular mass between sensitive and resistant cells were due to differences in N-glycosylation, we enzymatically deMLN4924 Metabolic Enzyme/Protease inhibitor glycosylated IGF1R and IR with PNGage F, which removed all types of N-linked glycans. Treatment with PNGage F increased the electrophoretic mobility of both IGF1Rb and IRb in all figitumumab-sensitive cells , indicating that IGF1Rb and IRb in the sensitive cells were mostly N-linked glycosylated. We next determined whether the variation of NLG of the IGF1Rb subunit could be another candidate biomarker for figitumumab sensitivity. To identify a specific NLG site within the IGF1Rb subunit, we used a combination of enzymatic deglycosylation and mass spectrometry analysis. After evaluating the samples from both sensitive and resistant cells, we identified site-specific glycosylation at Asn900 and Asn913 among five putative NLG sites of the IGF1Rb subunit.