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. 2018 May 9;8:7323. doi: 10.1038/s41598-018-25510-9

Figure 2.

Figure 2

Determination of IGF-1R sialylation levels using Lectin affinity assay: (A) Sialylation status of IGF-1R: Equal amount of cell lysates from vector control cells were treated with 100 mU of C. perfringens sialidase and untreated lysates from GNE knockdown cells were separated on 8% SDS-PAGE followed by immunoblotting with anti-α IGF-1R. (B) SNA Lectin affinity assay: Sialylated IGF-1R from GNE knockdown cells and scrambled shRNA vector control cell was pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. (C) Sialylated IGF-1R complexes from GNE mutant and pcDNA3 transfected vector control cells were pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. Sialylated IGF-1R complexes from 100 mU neuraminidase treated vector control cells that were pulled down using biotin labelled SNA/streptavidin-coupled agarose served as negative control in this experiment. Equal amount of protein was used as input lysates. (D–F) are showing representative densitometry graphs of (A–C), respectively, normalized to vector control.