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. 2013 Oct 10;4(10):e852. doi: 10.1038/cddis.2013.381

Figure 5.

Figure 5

Phosphorylation of amino-acid residues within c-Jun N-terminal domain. (a) Western blot analysis of HEK-293 cells transfected with DNA constructs expressing either HA-tagged c-Jun-wt or Ha-tagged c-JunA95, as indicated. After transfection, cells were treated either without or with Anisomycin (100 ng/ml) for indicated time-periods. c-Jun N-terminal phosphorylation was determined by using the indicated antibodies. (b) Lysates from anisomycin-treated c-Jun-wt-transfected HEK-T cells were immunoprecipited using the c-total c-Jun antibody, then aliquots from c-Jun immunocomplexes were analyzed by western blot using either c-Jun total antibodies (left panel) or c-Jun-p91/93 antibodies (right panel). (c) MALDI MS/MS spectra of the [M+H]+ from segments 82–97 carrying three phosphate groups at T91, T93 and T95. (d) MALDI MS/MS spectra of the [M+Na]+ from segments 62–70 carrying one phosphate group at S63. The a, b and y ions are labeled in each spectrum, as well as b or y ions corresponding to the neutral loss of phosphoric acid (−98)