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. 2017 Jul 17;7:5622. doi: 10.1038/s41598-017-06012-6

Figure 3.

Figure 3

CDK5 phosphorylates XBP1s at Ser61. (A) Mass spectrometry showed that CDK5 phosphorylated XBP1s at Ser61. The peptide RLTHLSPEEKA, which contained the XBP1s Ser61 residue, was synthesized in vitro. (B) CDK5/p25 phosphorylated XBP1s at Ser61 in an in vitro kinase assay. Purified glutathione-S-transferase (GST)-XBP1s WT or S61A fusion proteins were mixed with active CDK5/p25, and the phosphorylation signal was analyzed using a Phos S/T antibody. (C) Statistical analysis of the expression of phosphorylated GST-XBP1s WT or S61A fusion proteins. (D) The phosphorylation signal of GST-XBP1s WT or S61A fusion proteins was analyzed by Phos-tag SDS-PAGE gels. (E) CDK5/p35 phosphorylated XBP1s at Ser61 in HEK293 cells. HEK293 cells were co-transfected with HA-CDK5/Myc-p35 and GFP-XBP1s-WT or GFP-XBP1s-S61A plasmids, and then collected and immunoprecipitated with a Phos S/T antibody 24 h after transfection. (F) The transmembrane peptide Myr-XBP1, which contained the XBP1s Ser61 residue, had a protective effect on MPP+-induced cell death. Primary cultured neurons were pretreated with XBP1 peptide or scrambled peptide (0.1 μM, 1 μM, 10 μM, 30 min), and then treated with MPP+ (250 μM, 24 h). Cell survival was visualized by MTT assay. Data are mean ± s.d. of n = 3 independent experiments. Significance was determined by unpaired Student’s t test (C and F). *P < 0.05, **P < 0.01, ****P < 0.0001.