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1.
Fig. 2.

Fig. 2. From: Specific inactivation of two immunomodulatory SIGLEC genes during human evolution.

Cell-type specific expression of Siglec-13 and -17. (A) Chimpanzee and human peripheral blood mononuclear cells (PBMCs) were labeled with monoclonal anti–Siglec-13 mouse IgG. Colabeling for CD14 (a marker for PBMCs) showed specific expression in chimpanzee (Upper), but not in human (Lower) monocytes. (B) Transcript expression profile of human SIGLEC17P in 84 human tissues and cell lines acquired from BioGPS with probe gnf1h07492_at. The list of tissues and cell types studied can be found in the SI Appendix. (C) Quantitative RT-PCR for SIGLEC17P expression in human NK cells. Human T cells, whole-blood PBMCs, and whole-blood PBMCs depleted of T cells were used as controls. Results were normalized to GAPDH expression.

Xiaoxia Wang, et al. Proc Natl Acad Sci U S A. 2012 June 19;109(25):9935-9940.
2.
Fig. 4.

Fig. 4. From: Specific inactivation of two immunomodulatory SIGLEC genes during human evolution.

Chimpanzee Siglec-13 or resurrected Siglec-17 interacts selectively with bacterial pathogens. (A) Chimpanzee Siglec-13-Fc or resurrected human Siglec-17-Fc (with Arg) chimeras were immobilized to ELISA wells by protein A, and binding of FITC-labeled sialylated strains GBS A909 (serotype Ia) or E. coli K1 RS218 (StrR) was studied. Negative controls were L. lactis and nonencapsulated laboratory E. coli K-12 strain DH5α. (B) Binding of E. coli K1 strain RS218 (StrR), isogenic Sia-deficient E. coli K1 ΔneuDB, or E. coli K1 ΔneuDB pretreated with trypsin was studied as in A. (C) Binding of GBS A909 serotype Ia, isogenic Sia-deficient GBS ΔneuA, or GBSΔneuA pretreated with trypsin was studied as in A. StrainsΔBac (lacking β-protein) or the plasmid complemented mutant ΔBac + pBac were also studied. All values are means from three independent experiments ± SD.

Xiaoxia Wang, et al. Proc Natl Acad Sci U S A. 2012 June 19;109(25):9935-9940.
3.
Fig. 1.

Fig. 1. From: Specific inactivation of two immunomodulatory SIGLEC genes during human evolution.

Inactivation of two SIGLEC genes during hominin evolution. (A) Comparison of genomic structure surrounding the SIGLEC13 locus among humans and other primates. Coding regions are represented by shaded boxes, and Alu elements are represented by triangles. Names of Alu subfamilies are shown. Open boxes and ellipses indicate LINE (long interspersed element) and LTR (long terminal repeat) elements, respectively. The dotted line indicates a sequence gap. (B) DNA sequence alignment using Clustal W in MEGA4 shows the human-specific loss of G in SIGLEC17P (highlighted in gray). (C) Reconstructed neighbor-joining (NJ) tree of SIGLEC17P and SIGLEC3 among primates. Bootstrap values of 1,000 replicates are shown on internal branches. MEGA4 was used for NJ tree reconstruction and bootstrap analysis.

Xiaoxia Wang, et al. Proc Natl Acad Sci U S A. 2012 June 19;109(25):9935-9940.
4.
Fig. 5.

Fig. 5. From: Specific inactivation of two immunomodulatory SIGLEC genes during human evolution.

Reduced intracellular TNF of Siglec-13–transfected RAW264.7 cells in response to bacterial pathogen infection; 500,000 cells semistably transfected with Siglec-13 or vector only and selected with G418 for 3 wk were seeded in a 12-well plate. The next day, 2 h before infection, cells were washed three times with HBSS, and regular culture medium without G418 added. Cells were infected with bacterial pathogens at an appropriate MOI for 1 h at 37 °C. The BD Fixation and Permeabilization Solution Kit (555028) with APC rat anti-mouse TNF-α was used to detect the intracellular TNF level using the recommended protocol. Cells without bacterial infection were used as controls. Cells transfected with pcDNA3.1 vector were used as a control. (A) Cells infected by E. coli K1 RS218 (StrR) at MOI = 0.6. (B) Cells infected by GBS A909 (serotype Ia) at MOI = 0.1.

Xiaoxia Wang, et al. Proc Natl Acad Sci U S A. 2012 June 19;109(25):9935-9940.
5.
Fig. 3.

Fig. 3. From: Specific inactivation of two immunomodulatory SIGLEC genes during human evolution.

Importance of DAP12 for optimal surface expression of Siglec-13 and -17. (A) 293T cells were transiently transfected with a Siglec-17 cDNA in pcDNA3.1 with or without cotransfection with FLAG-tagged DAP12. Human CD33-transfected cells were used positive controls for detection by a rabbit anti-human CD33 antibody, which partially cross-reacts with human Siglec-17. Cells cotransfected with pcDNA3.1 and DAP12 were used as a negative control. Fluorescence was measured after staining with rabbit anti-human CD33 and then Alexa Fluor 647 donkey anti-rabbit IgG. The Siglec-17TMmutant in pcDNA3.1 was made from Siglec-17–pcDNA3.1 by introducing a K253A mutation. Cotransfected pIRES2-EGFP (Clontech) was used to gate positively transfected cells. (B) The 293T cells were transiently transfected with a Siglec-13 cDNA in pcDNA3.1 with or without cotransfection with FLAG-tagged DAP12. Controls were as in A. Fluorescence was measured after staining with mouse anti–Siglec-13 and then Alexa Fluor 647 goat anti-mouse IgG. The cSiglec-13TMmutant in pcDNA3.1 was made from cSiglec-13–pcDNA3.1 by introducing a K352A mutation. Cotransfected pIRES2-EGFP (Clontech) was used to gate positively transfected cells. (C and D) 293T cells transiently transfected with cDNAs for chimpanzee Siglec-13 (C) or human Siglec-17 (D) with/without DAP12 were lysed. M2 agarose beads were used to pull down FLAG tagged DAP12. Mouse anti-Siglec-13 or rabbit anti-human CD33 (which cross reacts with Siglc-17) were used in Western blots followed by HRP conjugated secondary antibodies, as shown in C and D, respectively. The upper band on the coimmunoprecipitation is nonspecific because of the use of M2 beads carrying a mouse antibody (C). M indicates All Blue protein standard (BIO-RAD).

Xiaoxia Wang, et al. Proc Natl Acad Sci U S A. 2012 June 19;109(25):9935-9940.

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