NDPK-B is Degraded by Ubiquitin Proteasome Pathway in HeLa cells

Abstract

Aim This topic clarifies that the NDPK-B is degraded by ubiquitin proteasome pathway in HeLa cells. Experimental The HeLa cells were treated with protein synthesis inhibitor Cycloheximide (CHX) in combination with proteasome inhibitor MG132 or lysosomal inhibitor leupeptin for different time, immunoblotting was performed to measure the level of NDPK-B and GAPDH. Ub-HA was overexpressed in HeLa cells, and immunoblotting was used to analyze the expression of Ub, NDPK-B and GAPDH. Result and discussion HeLa cells were treated with CHX individually; it showed that the level of NDPK-B was decreasing. So the degradation of NDPK-B in HeLa cells was definitive. After the joint treatment of leupeptin and CHX, the level of NDPK-Bin HeLa cells remained decreasing. So we suspected that NDPK-B was not likely to be degraded by the lysosomal pathway. Furthermore, HeLa cells were treated with CHX and MG132 together?we found that the level of NDPK-B remained unchanged. Therefore, we initially confirmed that NDPK-B was degraded by ubiquitin proteasome pathway in HeLa cells. Ubiquitination is a necessary condition for degradation of proteins through the ubiquitin proteasome pathway. Overexpression of Ub-HAin HeLa cells can cause degradation of NDPK-B. In addition, immunoprecipitation was performed to study the relationship between NDPK-B and Ub.The result showed that NDPK-B was ubiquitinated in HeLa cells. These results fully prove that NDPK-B is degraded by the ubiquitin proteasome pathway. Conclusion It is obvious that the NDPK-B is degraded by ubiquitin proteasome pathway in HeLa cells.