Anti-mouse CXCL10 antibody was purchased from R&D System, Inc

current EBV reactivation. The differentially expressed 618 probe sets were categorized by DAVID to assign annotation groups, based on gene ontologies. Gene Ontology/pathway Analysis The Database for Annotation, Visualization, and Integrated Discovery Bioinformatics Resource 6.7 from the National Institute of Allergy and Infectious Diseases was applied to generate clusters of functionally related genes. The Functional Annotation Clustering tool was used to generate clusters of overrepresented Gene Ontology terms. For categorizing genes according to the criteria of ten hallmarks of cancers, the QuickGO of the Gene Ontology Annotation Database was applied to determine the function and process of examined genes. PCR for genes of interest. Document S1 Statistical Analysis Differences between multiple groups were analyzed by oneway ANOVA with Tukey’s method for pairwise comparisons. T-test was used for Sodium laureth sulfate site comparisons of two groups. All statistical tests were two sided, and p,0.05 was considered to be statistically significant. List of cited references in S5. tory segment and an overlapping CaM-binding segment . In particular, three truncation forms of CaMKI exhibit distinct characteristics in basal kinase activity and responsiveness to Ca2+/CaM: the truncate encompassing residues 1321 which contains both the autoinhibitory segment and the CaM-binding segment displays properties similar to those of the full-length protein; the truncate encompassing residues 1314 which contains the autoinhibitory segment has a substantially lower kinase activity than the full-length kinase although it can bind Ca2+/CaM; and the truncate encompassing residues 1293 which excludes the regulatory region is constitutively active and exhibits an activity comparable to that of the fulllength kinase. The crystal structure of the apo rat CaMKI320 has been determined, leading to the proposal that the regulatory region of CaMKI inhibits the kinase activity by interacting with the N-terminal lobe and hence occluding the nucleotide-binding site and restraining the kinase in an inactive conformation. However, the regulation mechanism of CaMKI remains elusive. Here we report four crystal structures of three truncation variants of human CaMKIa, namely CaMKI320, CaMKI315, and CaMKI293, which correspond to the aforementioned three truncation forms of CaMKI, respectively. The structural data reveal new insights into the regulation mechanism of CaMKI. Structures of Human CaMKIa Materials and Methods Cloning, expression, and purification of CaMKI proteins The cDNA fragments encoding different human CaMKIa truncates were inserted into the BamHI and SalI restriction sites of the pGEX4T-1 expression plasmid which attaches a GST tag at the N-terminus of the protein. The plasmids were transformed into E. coli BL21 strain, and the transformed cells were grown in LB medium at 37uC in the presence of 50 mg/ml ampicillin until OD600 reached 0.8 and then induced with 0.5 mM IPTG for 12 hours at 16uC. The cells were harvested and lysed by sonication in a lysis buffer. Protein purification was carried out by affinity chromatography using a GSTrap FF 5 ml column. Cleavage of the fusion proteins with thrombin protease on column was conducted at 16uC for 24 hours. The elution fractions were further purified by gel filtration using a Superdex 200 16/60 column. The target proteins were concentrated to about 10 mg/ml with Centricon-10 for structural studies. Results Overall structure To explore the m