Journal: The Journal of Physiological Sciences : JPS

Article Title: Transient receptor potential cation 3 channel regulates melanoma proliferation and migration

doi: 10.1007/s12576-016-0480-1

Figure Lengend Snippet: TRPC3 expression and SOCE in melanoma. a Representative images of immunohistochemical staining of HE, MART1, and TRPC3 in a melanoma primary tissue microarray (stage II) (original magnification, b ×200). The calibration bars represent 200 µm. b mRNA expression in various melanoma cell lines. SK-Mel-2 is a skin metastasis melanoma cell line with NRAS mutation, SK-Mel-24 is a lymph node metastasis cell line with BRAFV600E mutation, SK-Mel-187 is also a lymph node metastasis cell line, C8161 is a metastasis cell line with wild-type BRAF, and HEMA-LP is a skin melanocyte cell line. TRPC3 mRNA is widely expressed in human melanoma cell lines, independently of BRAF mutation. c Immunoprecipitation for TRPC3 and STIM1 was performed. d Cytosolic Ca2+ level in C8161 cells is shown as mean ± SD (n = 8–9). SOCE was examined in the presence or absence of DMSO (vehicle control 1 µM) or Pyr3 (1 µM) in C8161. Pyr3 inhibited SOCE in C8161 cells. Pyr3 was added after the addition of Ca2+. The Ca2+ signal was immediately decreased, indicating that Pyr3 inhibits SOCE in melanoma cells. Data in each panel are averages of eight or nine cells

Article Snippet: C8161 cells treated with DMSO or Pyr3 (10 μM) for 15 min was subjected to protein phosphorylation microarray assay using a commercial kit (Cancer Signaling Phospho-Antibody Array; Full Moon BioSystems, Inc.).

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray, Mutagenesis, Immunoprecipitation, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: Transient receptor potential cation 3 channel regulates melanoma proliferation and migration

doi: 10.1007/s12576-016-0480-1

Figure Lengend Snippet: Pyr3 inhibits phosphorylation of STAT5 and Akt. a Representative images of Akt phosphorylation are shown. Densitometric analyses (bar graph) of Western blots showed that phosphorylation of Akt was inhibited by TRPC3 inhibitor, Pyr3 (n = 4, **p < 0.01, ns not significant). b Protein phosphorylation microarray analysis in the presence of Pyr3. C8161 cells were incubated with DMSO (vehicle control) or Pyr3 (10 μM) for 15 min. The Y-axis shows the signal ratio of phosphorylated to non-phosphorylated protein in the presence of Pyr3 as a percentage of that of the DMSO control. c Representative images of STAT5 phosphorylation. Densitometric analyses (bar graph) of Western blots showed that phosphorylation of STAT5 was inhibited by Pyr3 (n = 4, *p < 0.05, ns not significant)

Article Snippet: C8161 cells treated with DMSO or Pyr3 (10 μM) for 15 min was subjected to protein phosphorylation microarray assay using a commercial kit (Cancer Signaling Phospho-Antibody Array; Full Moon BioSystems, Inc.).

Techniques: Phospho-proteomics, Western Blot, Microarray, Incubation, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

doi: 10.4049/jimmunol.1400663

Figure Lengend Snippet: (A) IgG subtype analysis. Antibodies were engendered in C57Bl/6 mice against nickel column-purified VACV IMV membrane protein, WR101/H3LΔTM that has been adjuvanted in CpG/ISCOMs or alum, or in PBS alone as a control. Sera were obtained after 14 days and probed against VACV proteome microarrays on to which 8 two-fold serial dilutions of purified WR101/H3L were printed. Specific reactivity to purified WR101/H3L was visualized using fluorescently-tagged secondary antibodies to IgG, IgG1 and IgG2c and signal intensities quantified in a confocal laser scanner; data for a single concentration of printed antigen is shown. (B) Relative proportions of IgG1 and IgG2c derived from data shown in (A). The IgG2a proportion of the total signal is shown above the zero line, and the IgG1 proportion shown below. The IgG response is polarized according to adjuvant. (C) Protection of B6 mice against intranasal (i.n.) challenge of VACV-WR using adjuvanted WR101/H3LΔTM and WR101/H3L. CpG/ISCOMs reduce weight loss and promote recovery compared to alum or PBS. (D) and (E) correlations between nadir body weight (expressed as percentage of original body weight) and titer of IgG2c and IgG1, respectively. Titer was defined from the WR101/H3L titration series on the array at the lowest concentration to give a signal intensity >2000. Liner regression was used to generate the trend lines.

Article Snippet: We thank Douglas Molina, Andy Teng and the microarray printing team at Antigen Discovery Inc., for producing the VACV-WR proteome arrays used in this study.

Techniques: Nickel Column, Purification, Membrane, Control, Concentration Assay, Derivative Assay, Adjuvant, Titration

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

doi: 10.4049/jimmunol.1400663

Figure Lengend Snippet: Groups of five B6 mice were administered five different VACV antigens in CpG/ISCOM adjuvant. WR113/D8L was purified by conventional nickel-chelate chromatography optimized for this protein, whereas the remaining four antigens were all expressed and purified as SDS-IBs using a generic protocol. (A) Antibody profiles on d14 prior to VACV-WR i.n. challenge determined using VACV-WR proteome arrays. (B) Protection studies of mice against i.n. challenge of VACV-WR. (C) Protection studies of mice administered SDS-IBs of four different negative control antigens from B. melitensis, two positive control antigens from VACV-WR, or empty vector controls.

Article Snippet: We thank Douglas Molina, Andy Teng and the microarray printing team at Antigen Discovery Inc., for producing the VACV-WR proteome arrays used in this study.

Techniques: Adjuvant, Purification, Chromatography, Negative Control, Positive Control, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

doi: 10.4049/jimmunol.1400663

Figure Lengend Snippet: Spleen cells from mice administered four different individual VACV-WR proteins adjuvanted in CpG/ISCOMs and challenged with VACV-WR via i.n. route (shown in Figs. 2 and S1) were screened against the full SDS-IB VACV-WR proteome (n=220 proteins) in IFNγ ELISPOTs. Black symbols are numbers of spot-forming cells from mice administered WR070/I1L, WR113/D8L, WR118/D13L and WR148/− in CpG/ISCOM adjuvant (panels A, B, C and D, respectively). Grey symbols are responses by unimmunized/non-challenged control mice and are repeated in each panel for comparison. Eight replicate wells containing inclusion body preps made from bacteria transformed with empty expression vector are shown, right (EV).The hashed line corresponds to the average +3SD of the response to all the antigens by the uninfected mice.

Article Snippet: We thank Douglas Molina, Andy Teng and the microarray printing team at Antigen Discovery Inc., for producing the VACV-WR proteome arrays used in this study.

Techniques: Adjuvant, Control, Comparison, Bacteria, Transformation Assay, Expressing, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

doi: 10.4049/jimmunol.1400663

Figure Lengend Snippet: C57Bl/6 mice were administered VACV-WR i.p. (1E+5) on d0 and challenged by the i.n. route (2E+6) on d14. No weight loss was seen during this time. Spleen cells from three mice were pooled on days 8, 13 and 20 post-challenge and subjected to screening against the full SDS-IB VACV-WR proteome (n=220 proteins) in IFN-γ ELISPOTs, at 5×105 cells/well (panels A, B and C, respectively). Four immunodominant antigens are highlighted for orientation. Cells were also screened against 24 control antigens comprising solubilized SDS-IBs produced from Plasmodium falciparum (30) and Francisella tuarensis (31) expression libraries, and 7 empty wells that contained spleen cells with no antigens. One well contained 1μg/ml Concanavalin A as a positive control (not shown), which usually gave >600 spots/well and was defined by the plate counter software as too numerous to count. Hashed lines represent a cut-off defined as the means+2SD of the response to control antigens. D, uninfected mice. Pairwise comparisons of profiles were performed by scatter plots: E, d8 v. d13; F, d13 v. d20; G, d8 v. d20.

Article Snippet: We thank Douglas Molina, Andy Teng and the microarray printing team at Antigen Discovery Inc., for producing the VACV-WR proteome arrays used in this study.

Techniques: Control, Produced, Expressing, Positive Control, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

doi: 10.4049/jimmunol.1400663

Figure Lengend Snippet: A. Scatter plots of IgG signals by microarray vs. corresponding T cell responses from representative IFNγ ELISPOT. Cut-offs (hashed lines); for T cells defined as mean+2.5SD of control antigens (n=35; SDS-IBs proteome n = 225); antibody targets antigens defined as described in Table 2 (n=26; proteome on array n=194). Different temporal expressions indicated by symbols, as defined by Yang et al (32) using cluster analysis of viral mRNAs at 0.5, 1, 2 and 4h infection time points: E1.1=early subcluster 1; E1.2=early subcluster 2; PR=post-replicative. B. Bar charts of antibody signals (average of 6 mice) and T cells (averages of spot-forming cells/106 on d8 and d13, as shown in Fig 4). The first 65 antigens are ranked by antibody signal and thereafter by T cell response. C. Pie charts showing proportions of reactive antibody and CD4 T cell targets, compared to the whole VACV-WR proteome. Each protein was classified into a functional category according to Yang et al (2010) (32). Note: most of the pseudogenes were omitted from the arrays, so the proteome pie charts for IgG and for the T cell screens differ slightly.

Article Snippet: We thank Douglas Molina, Andy Teng and the microarray printing team at Antigen Discovery Inc., for producing the VACV-WR proteome arrays used in this study.

Techniques: Microarray, Enzyme-linked Immunospot, Control, Infection, Functional Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

doi: 10.4049/jimmunol.1400663

Figure Lengend Snippet: C57Bl/6 mice were administered i.p. heat-inactivated VACV-WR (approx. 1×107 pfu equivalents/mouse) in alum on d0 and challenged i.n. on d22. Spleen cells from three mice were pooled on d10 and d29 and subjected to IFNγ ELISPOTs against the full SDS-IB VACV-WR proteome as described in Fig. 4. A, ELISPOT data from d10, after heat inactivated VACV-WR i.p. prime only. B, ELISPOT data from d29, after heat inactivated VACV-WR i.p. prime and ‘live’ VACV-WR i.n. boost. Four immunodominant antigens are highlighted for orientation. C. Pie charts showing proportions of reactive antibody and T cell targets on d29. Whole VACV-WR proteomes for comparison shown in Fig. 5.

Article Snippet: We thank Douglas Molina, Andy Teng and the microarray printing team at Antigen Discovery Inc., for producing the VACV-WR proteome arrays used in this study.

Techniques: Enzyme-linked Immunospot, Comparison