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. 2023 Sep 20;26(10):107919. doi: 10.1016/j.isci.2023.107919

Figure 6.

Figure 6

5M-8OH-Q inhibits both UGGT1 and UGGT2 in cellula

(A) ALG6−/−, ALG6/UGGT1−/− and ALG6/UGGT2−/− HEK293-6E cells were cultured and either not treated or treated with 1 mM 5M-8OH-Q to determine if the drug inhibits one or both of UGGT1 and UGGT2. After the cells were incubated with the inhibitor, they were lysed and split between a whole-cell lysate sample (20%, “WCL”) and a GST-CRT pull-down sample (60%, “CRT”), and resolved by 9% SDS-PAGE gel electrophoresis, before transferring the protein bands to a PVDF membrane. Imaged are immunoblots probed for IGF1R (UGGT1 substrate48) and HexB (UGGT2 substrate48). Glucosylation of human ProIGF1R and human ProHexB was observed in ALG6−/−, ALG6/UGGT2−/−, and ALG6−/−, ALG6/UGGT1−/− cell lines, respectively. Each data point represents three independent biological replicates. GAPDH was used as a loading control.

(B and C) Quantification of human ProIGF1R and human ProHexB glucosylation from (A) Percent glucosylation was calculated by dividing the normalized value from the CRT lane by the normalized WCL. The resulting value was multiplied by 100 to obtain percent glucosylation. Error bars represent the standard deviation. Statistical significance levels: ∗: p 0.05; ∗∗: p 0.01; ∗∗∗: p 0.001.