Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2000 Sep 18;150(6):1335–1348. doi: 10.1083/jcb.150.6.1335

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Functional activity of zebrafish p38 and MKK3. (A) Expression levels of zebrafish p38a, p38b, and MKK3 throughout development. RT-PCR for zebrafish p38a, p38b, and MKK3 cDNAs are shown. pBLSK, positive control for specific primer pairs for zebrafish p38a, p38b, and MKK3 cDNA clones. max, loading control. (B) Activity of zebrafish p38 and MKK3 mutants expressed in 293T cells. Western blot of cell lysates from 293T cells transfected with expression vectors for WT-p38a, DN-p38a, WT-p38b, DN-p38b, DN-MKK3, CA-MKK3, or WT-JNK and human CA-MKK4 are as indicated. Activation of WT-p38a was detected in 293T cells expressing WT-p38a and CA-MKK3 by the anti–dp-p38 antibody (anti–ACTIVE p38™ antibody; lane 1) or in the cells treated with UV light (lane 4). No activation of WT-p38a was detected in the cells overexpressing either DN-p38a (lane 2) or DN-MKK3 (lane 3). DN-p38a and DN-MKK also suppressed UV-induced activation of WT-p38a (lanes 5 and 6). Activation of WT-p38b was also detected in 293T cells expressing WT-p38b and CA-MKK3 (lane 7) or in the cells treated with UV light (lane 10), but not in the cells expressing DN-p38b or DN-MKK3 (lanes 8, 9, 11, and 12). p38 (lanes 1–12), the amount of WT-p38a and WT-p38b loaded that was detected with the anti-myc antibody. Myc-tagged WT-JNK, which was coexpressed with human CA-MKK4 in 293T cells, did not react with the anti–dp-p38 antibody (bottom panel, left). Endogenous p38 in 293T cells expressing CA-MKK3 reacted with the anti–dp-p38 antibody, but not with anti-myc antibody (bottom panel, right). (C) Activation of ATF2 in cells expressing MKK3. 293T cells were transfected with the GAL4-ATF2 (pFA-ATF2) expression vector and GAL4-dependent luciferase reporter construct together with the expression vectors for WT-p38a, DN-p38a, CA-MKK3, or DN-MKK3 constructs, or with the empty vector to control for endogenous p38 activity. Luciferase activity was measured in cell extracts and normalized against β-galactosidase activity as an internal control. The relative value of the luciferase activity was plotted. Error bars represent the SD (n = 3). (D) Endogenous p38 activation in zebrafish embryos. Embryos were injected with RNA (125 pg) for DN-MKK3, DN-p38a, WT-p38a as indicated. Protein extracts of the embryos at the early blastula stage were immunoblotted with anti–ACTIVE p38™ antibody. Amounts of endogenous p38, DN-p38a, or WT-p38a were detected by anti-p38 antibody (bottom).