Journal: PLoS ONE

Article Title: A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

doi: 10.1371/journal.pone.0136888

Figure Lengend Snippet: (A) The sequences of SP27 (Biotin- SA SPHHHSRLRSES GG ) and SP27’ (Biotin-SPHHHSRLRSES SAGG ). Underlined letters represent nonspecific flanking amino acids. (B) Both SP27 and SP27’ bind to anti-PGN mAb in a dose-dependent manner. (C) Both SP27 and SP27’ (100 μg/ml) specifically bind to anti-PGN mAb, but not to anti-LTA mAb. For ELISA assays in panel B and C, anti-PGN mAb or anti-LTA mAb was used to coat the wells at a concentration of 5 μg/ml, SP27, SP27’ or non-specific peptide L2 (in panel B, sequence: Biotin-HSGHWDFRQWWQPSGG) was then added at indicated concentrations and incubated at 37°C for 40 min, followed by detection with HRP-labeled streptavidin. (D) The structure diagram of MAP27. MAP27 was synthesized in a tetra-branched form that contains four copies of a sequence (SASPHHHSRLRSESGG) that mimics PGN epitope. (E) MAP27 binds to anti-PGN mAb specifically. (F) MAP27 binds to anti- S . aureus polyclonal antibodies specifically. For ELISA assays in panel E and F, MAP27 or MAPctrl was used to coat the wells of a microplate. Anti-PGN mAb or anti- S . aureus polyclonal antibodies was added, followed by detection with HRP-labeled antibodies. The absorbance was measured at OD 450nm . The results are shown as means ±SEM. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Briefly, 96-well plates were coated with 5 μg/ml monoclonal antibody (mAb) against PGN (AbD Serotec, MCA5792, clone No.11-232.3, isotype: mouse IgG3) or mAb against Lipoteichoic acid (LTA, Thermo scientific, MA1-7401, clone No. G35C, isotype: mouse IgG1) at 4°C for overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Sequencing, Incubation, Labeling, Synthesized

Journal: PLoS ONE

Article Title: A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

doi: 10.1371/journal.pone.0136888

Figure Lengend Snippet: Five days after the last boost with heat-inactivated S . aureus , all of the mice were infected with S . aureus (2×10 7 CFU/mouse). Spleen cells (n = 5 mice per group) were harvested three days post infection and incubated with cocktail (including PMA and ionomycin) and BFA for 6 h. Cells were then fixed, permeabilized and stained with FITC-anti CD3, APC-anti CD4, PE-anti IFN-γ and PE-anti IL-17A mAb, respectively. Intracellular cytokine analysis was performed by flow cytometry. All lymphocytes were first gated based on FSC and SSC. CD3 + lymphocytes were then further gated from the lymphocyte cluster according to the fluorescence signal intensities. The results were presented as mean±SEM. (A) The percentage of IFN-γ + CD3 + CD4 + cells. (B) The percentage of IFN-γ + CD3 + CD4 - cells. (C) Representative flow cytometry plots showing the percentage of IFN-γ + CD3 + cells in spleen. (D) The percentage of IL-17A + CD3 + CD4 + cells. (E) The percentage of IL-17A + CD3 + CD4 - cells. (F) Representative flow cytometry plots showing the percentage of IL-17A + CD3 + cells in spleen. * P<0.05. ns: not significantly different between two groups (p>0.05).

Article Snippet: Briefly, 96-well plates were coated with 5 μg/ml monoclonal antibody (mAb) against PGN (AbD Serotec, MCA5792, clone No.11-232.3, isotype: mouse IgG3) or mAb against Lipoteichoic acid (LTA, Thermo scientific, MA1-7401, clone No. G35C, isotype: mouse IgG1) at 4°C for overnight.

Techniques: Infection, Incubation, Staining, Flow Cytometry, Fluorescence

Journal: Cancers

Article Title: Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

doi: 10.3390/cancers13215460

Figure Lengend Snippet: EZRIN and ARP2/3 activity are required downstream of RAC1 to sustain HA-NIS PM residency. HA-NIS-TPC1 cells were transfected with either ( A ) GFP-RAC1-L61 or a siRNA against EZRIN, or both; ( B ) with CFP-EZRIN in the presence or absence of the RAC1 inhibitor EHT1864 (50 µM for 1 h); ( C ) with either mock (siLUC control) or siARP3, and treated or not with the ARP2/3 complex inhibitor CK666 (100 µM for 1 h); or ( D ) with either empty vector (Empty) or GFP-RAC1-L61, and either mock (siLUC control) or the siARP3, as indicated, and analyzed by surface protein biotinylation. Surface fractions and whole-cell lysates (WCL) were then analyzed by WB, as indicated. The ‘no Biotin’ condition corresponds to cells not incubated with biotin, and was used to control the specificity of biotinylated protein pull-downs. PCNA expression served as both the loading (WCL) and intracellular protein contamination control (Surface pool). GLUT-1 served as the positive loading control for the cell-surface protein extracts. The WB bands were quantified by densitometric analysis using ImageJ software. The plotted values correspond to the means ± SEM of at least three independent assays. One-way ANOVA analysis detected the significant differences between the treatments [F = 23.65 and p < 0.001 for ( A ); F = 17.86 and p < 0.001 for ( B ); F = 14.79 and p < 0.001 for ( C ); F = 18.26 and p < 0.001 for ( D )]. Post-hoc Tukey’s tests were used to identify significant variations relative to the control conditions or among the different treatments (the latter are indicated by horizontal lines) (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: The siRNA oligos against IQGAP1 (sc-35700), COPA (sc-43696), RAC1 (sc-36351), EZRIN (sc-35349), ARP3 (sc-29739), PAK1 (sc-29700) and PIP5K (sc-36232) were purchased from SCTB (Santa Cruz, CA, USA).

Techniques: Activity Assay, Transfection, Control, Plasmid Preparation, Incubation, Expressing, Software