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. 2022 Nov 25;13(3):1053–1070. doi: 10.1016/j.apsb.2022.11.025

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© 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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HBXIP enhances PKCβII-mediated NMHC-IIA phosphorylation fastening the interaction between HBXIP and NMHC-IIA. (A) Co-IP analysis of the phosphorylation level of Flag-NMHC-IIA was detected using an anti-phospho-serine antibody. pENTER-vector and pENTER-NMHC-IIA, along with GFP-HBXIP were transiently transfected into HEK-293T cells. (B) Co-IP analysis of the phosphorylation level of Flag-EATR. pCMV-EATR with GFP-HBXIP were transiently transfected into HEK-293T cells. (C) Co-IP analysis of the phosphorylation level of Flag-EATR. pCMV-EATR-WT, pCMV-EATR-S1916A, pCMV-EATR-S1943A and pCMV-EATR-DM together with pcDNA-HBXIP or pcDNA-vector were transiently transfected into HEK-293T cells. (D) Co-IP analysis of the interaction between Flag-S1916A/S1916D/DM and HBXIP. pCMV-EATR-S1916A, pCMV-EATR-S1916D and pCMV-EATR-DM together with pcDNA-HBXIP were transiently transfected into HEK-293T cells. (E) Schematic diagram of phosphorylation sites and kinases of NMHC-IIA. (F) Fold change in mRNA levels of potential kinases determined by RT-qPCR assays. pCMV-vector and pCMV-HBXIP or si-control and si-HBXIP were dose-dependently transfected into the cells. (G, H) Co-IP analysis of the interaction between endogenous or exogenous HBXIP and PKCβII in MCF-7 cells. (I) Co-IP analysis of the phosphorylation level of Flag-EATR. pCMV-EATR together with pcDNA-HBXIP or pcDNA-vector, along with si-control or si-PKCβII #1 were transiently transfected into HEK-293T cells. (J) Co-IP analysis of the phosphorylation level of Flag-EATR. Before treatment with 10 μmol/L LY333531 for 24 h, pCMV-EATR along with pcDNA-HBXIP or pcDNA-vector were transfected into HEK-293T cells. (K) The diagram shows the 3 separate segments of PKCβII constructed amino acids 2–673 (DN), 30–673 (ΔNPS) and 329–673 (CAT). (L) Co-IP analysis of interaction between EATR, PKCβII and HBXIP. pcDNA-PKCβII-WT, pcDNA-PKCβII-DN, pcDNA-PKCβII-ΔNPS and pcDNA-PKCβII-CAT along with pcDNA-HBXIP and pCMV-EATR were transiently transfected into HEK-293T cells. Three experiments with consistent results tendency were repeated.