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. 2021 Jan 13;59(1):74–86. doi: 10.1080/13880209.2020.1866024

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© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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graphic file with name IPHB_A_1866024_F0004a_B.jpg — Inhibitory effect of Sb-EE on AP-1 signalling enzymes. (A–C) RAW264.7 cells were treated with LPS (1 µg/mL) for the indicated time in the presence or absence of Sb-EE (200 μg/mL). Then, an immunoblotting assay was performed with antibodies for phospho- and total-forms of target proteins. The antibodies against p-ERK, ERK, p-JNK, JNK, p-p85 and p85 antibodies were used to detect the MAPKs (A). The antibodies against p-TAK1, TAK-1, p-MEK1/2, MEK1/2, p-MKK4/7, MKK4/7, p-MKK3/6 and MKK3/6 were used to detect the upstream enzymes of MAPKs (B, left and right panel). IRAK-1 and IRAK-4 antibodies were used to detect the initially activated enzymes in the AP-1 signalling pathway (C). (D) To examine the direct effects of Sb-EE on IRAK1 activity, an in vitro kinase assay was performed with purified IRAK1. ^##p < 0.01 compared to the normal group, *p < 0.05 and **p < 0.01 compared to the control group (LPS-alone group in (A), (B, left and right panel) and (C)).

graphic file with name IPHB_A_1866024_F0004b_B.jpg — Inhibitory effect of Sb-EE on AP-1 signalling enzymes. (A–C) RAW264.7 cells were treated with LPS (1 µg/mL) for the indicated time in the presence or absence of Sb-EE (200 μg/mL). Then, an immunoblotting assay was performed with antibodies for phospho- and total-forms of target proteins. The antibodies against p-ERK, ERK, p-JNK, JNK, p-p85 and p85 antibodies were used to detect the MAPKs (A). The antibodies against p-TAK1, TAK-1, p-MEK1/2, MEK1/2, p-MKK4/7, MKK4/7, p-MKK3/6 and MKK3/6 were used to detect the upstream enzymes of MAPKs (B, left and right panel). IRAK-1 and IRAK-4 antibodies were used to detect the initially activated enzymes in the AP-1 signalling pathway (C). (D) To examine the direct effects of Sb-EE on IRAK1 activity, an in vitro kinase assay was performed with purified IRAK1. ^##p < 0.01 compared to the normal group, *p < 0.05 and **p < 0.01 compared to the control group (LPS-alone group in (A), (B, left and right panel) and (C)).