Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A ) Expression of CD27 and IgD on CD19 + B cells. Right: Percentage of IgD + CD27 − naïve B cells and IgD − CD27 − DN B cells. ( B ) Expression of CD27 and CD38 on CD19 + B cells. Right: Percentages of CD27 hi CD38 hi ASCs and CD27 + CD38 − memory B cells. ( C ) Expression of CD138 on CD27 hi CD38 hi ASCs. Right: Percentage of CD138 + ASCs and CD138 − ASCs. ( D ) Expression of CD11c and CD21 on CD19 + B cells. Right: Percentage of CD11c + CD21 − CD19 + B cells. [(A) to (D)] HC ( n = 63), irAE ( n = 33), RAC ( n = 46), and ICI ( n = 20). ( E to G ) GSEA was performed on the B cells between irAE and ICI. (E) Significantly enriched pathways in B cells from irAE and ICI. GSEA plots of IFN-α and IFN-γ response (F), and oxidative phosphorylation (G). ( H ) The volcano plots of the citrullinated or noncitrullinated relative IgG or IgM isotype autoantigen levels comparing RA versus HC, irAE versus ICI, irAE versus HC, or irAE versus RA. The autoantigens were labeled when P < 0.01. TNF-α reactivities resulted from the administration of anti–TNF-α therapy for the treatment of RA. ( I ) Immunoglobulin isotype levels in the plasma were measured by multiplex assay. HC ( n = 22), irAE ( n = 34), RAC ( n = 46), and ICI ( n = 26). ( J ) CXCL13 levels in the plasma were measured by enzyme-linked immunosorbent assay (ELISA). HC ( n = 20), irAE ( n = 34) RAC ( n = 47), and ICI ( n = 23). ( K ) B-cell activating factor (BAFF) levels in the plasma were measured by multiplex assay. HC ( n = 21), irAE ( n = 34), RAC ( n = 47), and ICI ( n = 18). Data in graphs represent mean ± SEM. Significance was tested by one-way ANOVA [(A) to (D) and (I) to (K)] and logistic regression (H). [(A) to (D) and (F) to (K)] ICI, ICI control.

Article Snippet: For CXCL13, IL-21, and CX3CL1 measurements, the following kits were used: Human CXCL13/BLC/BCA-1 Quantikine Enzyme-Linked Immunosorbent Assay (ELISA) (R&D Systems, catalog no. DCX130), Human IL-21 DuoSet ELISA (R&D Systems, catalog no. DY8879-05), and Human CX3CL1/Fractalkine DuoSet ELISA (R&D Systems, DY365); all steps were performed according to the manufacturer’s instructions.

Techniques: Expressing, Phospho-proteomics, Immunopeptidomics, Labeling, Clinical Proteomics, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The inhibitory activities of Taiwanese local pomegranate fruit. The ethylacetate, butanol, and water layers extracted from pomegranate peel extract (PEP) were tested for the inhibitory efficacy to T24 ( A ) or J82 ( B ) cells using MTT assay as described in Materials and Methods. PEPE2 and PEPE3 ( C ) were also investigated for the inhibitory effectiveness to T24 or J82 cells. 0.1% ( v / v ) Dimethyl sulfoxide (DMSO)-treated urinary bladder urothelial carcinoma (UBUC) cells were regarded as the solvent control. Each MTT result was the typical data of at least three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: MTT Assay, Solvent, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The propidium iodide (PI) and PI/annexin V analyses of UBUC cells treated with PEPE2. The results of PI analyses were represented in ( A ) the T24 cells and ( B ) the J82 cells. The data of the PI/annexin V measurement were shown in ( C ) T24 cells and ( D ) J82 cells. Each flow cytometry figure was the typical result of three independent experiments. The diagram under the PI/annexin V panel was the results of the three independent experiments. The cell cycles and apoptosis detection of the T24 or J82 cells treated with PEPE2 were measured with PI and PI/annexin V analyses, respectively, using flow cytometry as described in the . The 0.1% ( v / v ) DMSO-treated UBUC cells were implemented as the vehicle control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Flow Cytometry, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The molecular mechanisms of apoptotic pathway evoked in PEPE2-incubated UBUC cells. T24 and J82 cells were treated with 50 and 20 μg/mL PEPE2 respectively. Then the protein levels of ( A ) pro-/cleaved caspase-3; ( B ) pro-/cleaved caspase-8, DR4 and DR5; ( C ) pro-/cleaved caspase-9, Bax and Bcl-2 and ( D ) Bip, VCP and pro- caspase-12 in PEPE2-treated T24 cells were measured using western immunoblotting as described in the supporting information. The 0.1% ( v / v ) DMSO-treated UBUC cells were used as the solvent control; ( E ) The proposed molecular apoptotic pathway provoked in the PEPE2-treated UBUC cells. The immunoblot in each figure was the representative result of at least three independent experiments. The diagram (ratio (mean ± standard deviation (S.D.)) under each immunoblot indicated the ratio of the normalized protein intensity (observed protein/actin) of PEPE2-treated cells at the indicated time interval, divided by that at the 0-h time point. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Incubation, Western Blot, Solvent, Control, Standard Deviation

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The inhibition of the xenografted UBUC growth in nude mice by the treatment of the EtOAc layer. T24 cells were injected s.c. into nude mice and were o.g. administrated with water (control), 2, 5, 10, or 100 mg/kg EtOAc layer as described in Materials and Methods. ( A ) The body weights of the extract-treated mice. *** p < 0.001, ** p < 0.005; ( B ) The effects of the EtOAc layer on tumor growth in the xenografted nude mice. The volumes of tumors from the extract-fed mice were compared to those of the water-fed mice. *** p < 0.001; ( C ) The tumor weight. The tumor weight was measured at the 10th week. *** p < 0.001; ( D ) The typical H/E images (400×) of tumor specimens dissected from xenografted nude mice; ( E ) The representative terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) images (400×) of tumor lesions from xenografted nude mice. H&E and TUNEL assays were performed as described in Material and Methods.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Inhibition, Injection, Control, End Labeling, TUNEL Assay

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Untreated hyperacute HIV, but not ART early-treated hyperacute HIV, is associated with elevation of plasma cytokines that have distinct kinetics. a Interferon gamma-induced protein 10 (IP-10/CXCL-10). b Monokine induced by gamma interferon (MIG/CXCL-9). c Monocyte chemoattractant protein 1 (MCP-1). d Interleukin 12 (IL-12). e Soluble IL-2 receptor (IL-2R). f Interleukin 8 (IL-8). g Interferon gamma (IFN-gamma). h Interleukin-1 receptor antagonist (IL-1RA). i B cell-activating factor (BAFF/BLYS/TNFSF13B). j Chemokine (C-X-C motif) ligand 13 (CXCL13). k Soluble CD14. l Interferon alpha (IFN-alpha). N = 12 for untreated hyperacute HIV-infected participants (except CXCL13 and BAFF with N = 10). N = 8 for ART early-treated hyperacute HIV-infected individuals (except CXCL13 and BAFF with N = 6 and IFN-alpha with N = 7). Cytokine levels for one of the untreated participants were measured 434 days instead of 238–263 days after the detection of viremia. Each symbol represents an individual participant. Except for IFN-alpha, red symbols show the plasma levels in untreated participants and blue symbols show the plasma levels in ART early-treated participants. Horizontal lines and error bars in the scatter plots represent the median and interquartile range. In l (IFN-alpha), every colored line represents a participant. Statistical test used: Wilcoxon matched-pairs signed-rank test. P values < 0.05 were considered significant. * P < 0.05, ** P < 0.01, *** P < 0.001. “Pre” refers to the pre-infection time point

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: CXCL13 is positively associated with delayed suppression of viremia in early-treated individuals. a Duration to viral suppression in days among early-treated participants. b Correlation between duration to viral suppression in days and viral load at the time of initiating ART in early-treated individuals. c Correlation between duration to viral suppression and plasma CXCL13 levels at 3 months. d Correlation between viral load at the time of initiating ART and plasma CXCL13 levels at 3 months. Each symbol represents an individual participant ( N = 6). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: The magnitude of plasma cytokines predicts CD4 + T cell and viral load dynamics in untreated hyperacute HIV infection. a Correlation between peak IFN-alpha and peak viremia. b Correlation between hyperacute soluble IL-2 receptor and peak viremia. c Correlation between hyperacute IL-1RA and viral load set point. d Correlation between hyperacute CXCL13 and nadir CD4 + T cell counts. e Correlation between hyperacute soluble IL-2 receptor and nadir CD4 + T cell counts. f Correlation between hyperacute IL-1RA and set point CD4 + T cell counts. Each symbol represents an individual participant ( N = 12 except CXCL13 with N = 10). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Plasma cytokines/chemokines are associated with reduced blood counts of lymphocytes, eosinophils, and basophils in untreated acutely HIV-infected patients. a Correlation between CXCL13 and total lymphocytes. b Correlation between CXCL13 and eosinophils. c Correlation between CXCL13 and basophils. d Correlation between MIG/CXCL9 and total lymphocytes. e Correlation between MIG/CXCL9 and eosinophils. f Correlation between MIG/CXCL9 and basophils. g Correlation between soluble IL-2 receptor and total lymphocytes. h Correlation between soluble IL-2 receptor and eosinophils. i Correlation between soluble IL-2 receptor and basophils. The measurements of cytokines and blood cell counts were in the hyperacute phase of HIV infection. Each symbol represents an individual participant ( N = 12 except CXCL13 ( a – c ) with N = 10). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Correlation network showing a summary of the relationships between cytokines, CD4 + T cell dynamics, viral load dynamics, Gag-driven viral replication capacity, and hematological parameters in untreated hyperacute HIV infection. Statistical test used: Spearman’s rank-order correlation. Red lines show significant positive correlations. Blue lines show significant inverse correlations. The width of the line indicates the strength of Spearman’s correlation coefficient (rho). Only correlations that have P < 0.05 are shown. Gag RC, Gag-driven viral replication capacity ( N = 12 except CXCL13 and BAFF with N = 10)

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Infection

Journal: BMC Physiology

Article Title: Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation

doi: 10.1186/1472-6793-7-4

Figure Lengend Snippet: A-E . PAR immunohistochemistry in J82 human urothelial carcinoma cell line. Representative photomicrograph obtained in J82 cell line stained with PAR specific antibodies. J82 cells were fixed and incubated with primary polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies: A = PAR-1 (1:20), B = PAR-2 (1:100), C = PAR-3 (1:5), and D = PAR-4 (1:50). Slides were washed and incubated with biotinylated secondary antibodies (Vector Labs), goat anti-rabbit (polyclonal antibodies). Orange dotted circles highlight some cells considered positives for the particular receptor. Original magnification was ×200. Figure 1E represents the average and SEM of number of PAR-positive cells as percent of the total cells per field.

Article Snippet: Human bladder carcinoma cell line J82 (HTB-1) were obtained from the American Tissue Culture Collection.

Techniques: Immunohistochemistry, Staining, Incubation, Plasmid Preparation

Journal: BMC Physiology

Article Title: Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation

doi: 10.1186/1472-6793-7-4

Figure Lengend Snippet: A-B . Polymerase Chain Reaction for detection of PARs message in J82 human urothelial carcinoma cell line. Figure 2A is a photomicrograph of the gel and Figure 2B represents the area under the curve as quantified using Image J software [74]. Primers used in this experiment are described in additional file (Table 1).

Article Snippet: Human bladder carcinoma cell line J82 (HTB-1) were obtained from the American Tissue Culture Collection.

Techniques: Polymerase Chain Reaction, Software

Journal: PLoS pathogens

Article Title: Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response.

doi: 10.1371/journal.ppat.1003141

Figure Lengend Snippet: Figure 2. Adhesion ability of AIEC and non-AIEC strains with regard to fimH clade and CEACAM6 expression. (A) Cell-associated bacteria were quantified using non differentiated T84 cells after a 3 H infection period, and results were analyzed with regard to fimH clade. (B) Cell- associated AIEC bacteria belonging to the fimH S70/N78 clade were quantified using undifferentiated and differentiated T84 cells after a 3 H infection period. (C) Western blot analysis of whole protein extracts from non-differentiated and differentiated T84 cells using anti-CEACAM6 and anti-GAPDH antibodies. (D) Cell-associated bacteria (AIEC and non-AIEC bacteria belonging to the fimH S70/N78 clade) were quantified using differentiated T84 cells after a 3 H infection period. (E) Yeast agglutination titer of AIEC and non-AIEC strains belonging to the fimH S70/N78 clade. (F) Transmission electron micrograph of negatively stained AIEC and non-AIEC bacteria belonging to the fimH S70/N78 clade, magnification 625 000. doi:10.1371/journal.ppat.1003141.g002

Article Snippet: Adhesion assay of human intestinal and bladder epithelial cells T84 cells (derived from human colorectal carcinoma) and T24 cells (human bladder epithelial cells) were purchased from ATCC and maintained in an atmosphere containing 5% CO2 at 37uC in appropriate medium.

Techniques: Expressing, Bacteria, Infection, Western Blot, Agglutination, Transmission Assay, Staining

Journal: PLoS pathogens

Article Title: Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response.

doi: 10.1371/journal.ppat.1003141

Figure Lengend Snippet: Figure 4. Impact of FimH amino acid substitutions on AIEC adhesion ability. Cell-associated bacteria were quantified using undifferentiated (A) or differentiated (B) T84 cells after a 3 H infection period. WT represents the original AIEC LF82 reference strain and DfimH represents the LF82- DfimH isogenic mutant. The other constructs were made in the LF82-DfimH isogenic mutant, in which various fimH variants were reintroduced at the fimH locus. Each value is the mean 6 SEM of at least four separate experiments (* P,0.05; ** P,0.001; *** P,0.001). (C) Relative ability to bind Man1 (1 M/3 M binding ratio) with regard to undifferentiated T84 index adhesion of the various fimH chromosomal mutants constructed. (D) Bacterial binding to 1M-BSA under various shear stresses (pN/m cm2) was evaluated by measuring bacterial accumulation over 3 min. doi:10.1371/journal.ppat.1003141.g004

Article Snippet: Adhesion assay of human intestinal and bladder epithelial cells T84 cells (derived from human colorectal carcinoma) and T24 cells (human bladder epithelial cells) were purchased from ATCC and maintained in an atmosphere containing 5% CO2 at 37uC in appropriate medium.

Techniques: Bacteria, Infection, Mutagenesis, Construct, Binding Assay, Shear

Journal: Cell death & disease

Article Title: The APC/C E3 ligase subunit ANAPC11 mediates FOXO3 protein degradation to promote cell proliferation and lymph node metastasis in urothelial bladder cancer.

doi: 10.1038/s41419-023-06000-x

Figure Lengend Snippet: Fig. 6 FOXO3 promotes the transcription of the CDK inhibitor p21 and GULP1. A The mRNA expression of 12 potential FOXO3 downstream effectors when FOXO3 was silenced in UBC cells, measured by RT–PCR. B The mRNA expression of GULP1 and p21 in T24 and UM-UC-3 cells overexpressing FOXO3, measured by RT-PCR. C, D Correlation analysis of CDKN1A and FOXO3 mRNA (C) and correlation analysis of GULP1 and FOXO3 mRNA (D) in TCGA-BLCA. E Enrichment of the CDKN1A promoter with anti-FOXO3 or IgG measured by ChIP followed by RT-PCR. F Luciferase reporter assays in 293 T to evaluate the regulation of FOXO3 expression on CDKN1A transcriptional activity. Firefly luciferase activity was normalized to Rluc intensity. G The prediction of FOXO3 binding motif via JASPAR. H Illustration of two potential binding sites of FOXO3 in GULP1 promoter. I Enrichment of two binding sites in GULP1 promoter with anti-FOXO3 or IgG measured by ChIP followed by RT- PCR. J The effect of FOXO3 expression on GULP1 transcriptional activity, evaluated by dual luciferase reporter assays. K, L Western blot analysis showed the expression of FOXO3, p21, and GULP1 when the FOXO3 level was downregulated (K) or upregulated (L) in UBC cells. Data are shown as mean ± SD. Unpaired, two-tailed student’s t-test, *P < 0.05, **P < 0.01.

Article Snippet: The human bladder urothelial carcinoma cell lines HT-1376, UM-UC-3, T24, J82, 5637, and RT4, the human immortalized uroepithelial cell line SV-HUC1 and the human embryonic kidney cell line 293 T were obtained from the American Type Culture Collection.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Binding Assay, Western Blot, Two Tailed Test