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. 2021 Jun 7;9:684885. doi: 10.3389/fcell.2021.684885

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Copyright © 2021 Gong, Wang, Wang, Hu, Li, Gao and Lin.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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CUL5–ASB6 complex promotes ubiquitination and degradation of p62. (A) IB analyses of WCL from HEK293T cells co-transfected with plasmids encoding HA-p62, ASB6 shRNAs, and GFP (as an internal transfection control). Tubulin served as a loading control. (B) HeLa cells infected with indicated shRNAs were harvested for IB analysis. Tubulin served as a loading control. The knockdown efficiency of ASB6 was examined by RT-PCR. GAPDH gene was detected as the internal control. (C) HEK293T cells were co-transfected with plasmids encoding HA-p62 and FLAG-ASB6 and were treated with different concentrations of MG132 (10, 20, and 30 μM) for 10 h before harvest for IB assay. Tubulin served as loading control. (D) IB analyses of HEK293T cells transfected with plasmids encoding HA-p62 and ASB6 mutants. Tubulin served as a loading control. (E,F) HEK293T cells were transfected with plasmids encoding HA-p62 and EV or FLAG-ASB6. Forty-eight hours after transfection, cells were treated with 50 μg/ml CHX and harvested at the indicated time for IB analysis. Tubulin served as loading control. Relative HA-p62 protein levels were quantified and normalized to Tubulin with ImageJ software. (G) IB analysis of HEK293T cells transfected with a plasmid encoding FLAG-ASB6. Tubulin served as a loading control. (H) HEK293T cells transfected with the indicated plasmids were treated with 10 μM MG132 for 10 h before harvest for Ni-NTA beads pull down and IB analysis.