PMC

MAP3K4 immunoprecipitations from MCF-7 breast cancer cells stimulated by HRG or sorbitol. A schematic representation of MAP3K4 domains identifying antibody epitopes directed against MAP3K4. The MAP3K4 (P) antibody recognizes an amino terminal region (amino acids 18–139) of MAP3K4 that is proline rich, while the MAP3K4 (A) antibody recognizes an internal region (amino acids 1,102–1,255) of MAP3K4 that is alanine rich (Panel A). Cells were stimulated with 10 nM HRG for 10 minutes or 0.3 M sorbitol for 30 minutes. Lysates were incubated with anti MAP3K4 (A) or anti MAP3K4 (P) antibody and precipitated with Protein A–Sepharose. Immunoprecipitated proteins were separated by SDS-PAGE. Proteins were transferred to nitrocellulose membrane and immunoblotted using the indicated antibodies (Panel B). MCF-7 cells were treated with 10 nM of HRG for the indicated time in minutes. Proteins were immunoprecipitated for MAP3K4 or MAP3K3, resolved by SDS-PAGE and immunoblotted as indicated (Panel C). Different areas of the nitrocellulose membrane are separated by white space. A vertical line separates groups belonging to different membranes.

AK094629 effects the expression of MAP3K4, but MAP3K4 has no effect on the expression of AK094629. (A) Expression of the MAP3K4 gene after knockdown of AK094629 was detected by RT-qPCR. (B and C) Expression of the MAP3K4 protein after knockdown of AK094629 was determined by (B) western blotting and (C) quantification. (D) Detection of AK094629 expression by RT-qPCR after knockdown of MAP3K4 using siMAP3K4-2 and siMAP3K4-3. *P<0.05 vs. NC group; ^#P<0.05 vs. NC+IL-1β group. MAP3K4, mitogen-activated protein kinase kinase kinase 4; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; NC, negative control; IL, interleukin.

(A–C) 293 cells were transfected with the indicated constructs. Western blots were probed with the indicated antibodies. (FL, Flag; KI, Kinase Inactive; WT, wild-type)
(A) Co-expression of wild-type HA-MAP3K4 and FL-HDAC6 increases the ubiquitination of HDAC6 but not MAP3K4. Samples were immunoprecipitated with the indicated antibody and probed for Myc-ubiquitin. Blots shown are representative of two independent experiments.
(B) Kinase inactive MAP3K4 does not promote ubiquitination of HDAC6. Samples were immunoprecipitated using anti-FL antibody and probed for Myc-ubiquitin. Data shown are representative of four independent experiments.
(C) Ubiquitination of wild-type and kinase inactive MAP3K4 is not influenced by HDAC6. Samples were immunoprecipitated with anti-HA antibody and probed for Myc-ubiquitin. Blots shown are representative of three independent experiments.
(D) The degradation rate of endogenous HDAC6 is reduced in the absence of MAP3K4 kinase activity. TS^WT or TS^KI4 cells were treated for the indicated number of hours with cycloheximide (CHX), an inhibitor of translation. Representative blots from three independent experiments are shown. Fold change relative to wild-type 0 hours was calculated by normalizing HDAC6 densitometry to total ERK.

Firefly luciferase activity of HIF1A and MAP3K4 targets in the presence or absence of miR-199b (control), showing slightly reduced luminosity, indicating HIF1A and MAP3K4 could be potential targets of miR-199b. Luciferase activity is normalized to Renilla activity and control (mean ± SEM).