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. Author manuscript; available in PMC: 2012 Sep 1.
Published in final edited form as: Nat Immunol. 2011 Aug 7;12(9):844–852. doi: 10.1038/ni.2080

Figure 6. Identification of S311 phosphorylation site of Act1 by MS.

Figure 6

Figure 6

A. MS/MS spectrum of the Act1 phosphopeptide (305VILNDSSpPQDEERPAQR322) precursor ions at m/z of 721.33 Da.

B. MS/MS spectrum of unmodified peptide of the same sequence (305VILNDSSPQDEERPAQR322) precursor ions at m/z of 695.01 Da.

C. Act1-deficient MEFs infected with either retroviral WT-mAct1 or Act1 mutant S11A were untreated or treated with IL-17 (50 ng/ml) for 0, 15 or 30 min, followed by immunoblot analysis with anti-Act1 and anti-Actin.

D. Act1-deficient MEFs infected with either retroviral WT-mAct1 or Act1 mutant S11A, followed by treatments with IL-17A (50 ng/ml) for 1 h and 3 h. CXCL1 and IL-6 mRNA was measured by real-time PCR.

E. Act1-MEFs infected either WT-mAct1 or Act1 mutant S11A were untreated or treated with IL-17 (50 ng/ml) for 0, 15, 30 or 60 min, followed by immunoblot analysis with antibodies against p-Erk, Erk1, p-IκB, IκB, p-Jnk, Jnk, p-p38, p38 and Actin.

F. HeLa tet-off cells were transfected with 1 µg of pTRE2 CXCL1Δ4 and 1 µg of Act1 or Act1 S311A mutant. The transfected cells were treated with dox (1 µg/ml) and incubated for the indicated times, followed by isolation of total RNA and RNA hybridization blot analysis for the determination of CXCL1 and GAPDH mRNA levels.

The data shown in this figure are representation of three independent experiments.