Journal: The Journal of Biological Chemistry

Article Title: Paraoxonase 3 functions as a chaperone to decrease functional expression of the epithelial sodium channel

doi: 10.1074/jbc.RA119.011789

Figure Lengend Snippet: Pon3 reduces ENaC surface and whole-cell expression in FRT cells. A, FRT cells were transiently transfected with three ENaC subunits, where only the α subunit had an N-terminal HA tag and a C-terminal V5 tag. Equal amounts of the mouse Pon3 plasmid or the pCMV6 vector were cotransfected. Surface ENaC was labeled with biotin and recovered with NeutrAvidin beads at 4 °C. Blots were probed for the α subunit, γ subunit, Pon3, or GAPDH in the biotinylated surface fraction and in 5% of the total whole-cell lysates. B, the abundance of the α subunit (full-length 95 kDa and cleaved 30 kDa, indicated by arrowheads) or the γ subunit (∼75 kDa) in whole-cell lysate was normalized to the loading control (GAPDH) and expressed as the percentage in cells transfected with ENaC alone (−Pon3). C, the abundance of the α subunit (full-length 95 kDa and cleaved 30 kDa) or the γ subunit (∼75 kDa) at the cell surface was normalized to whole-cell abundance of the α or γ subunit, respectively, and then expressed as the percentage in cells transfected with ENaC alone (−Pon3). D, as a control, FRT cells were cotransfected with HAαV5βγ and with either γ-glutamyl transferase (+γGT) or an equal amount of the pCDNA3 vector (−γGT). Whole-cell lysates were collected and probed for both αENaC and γGT (indicated by the arrow). E, the abundance of the α subunit (95 kDa and 30 kDa) was normalized to the loading control (GAPDH) and expressed as the percentage in cells transfected with ENaC alone (−γGT). Experiments were repeated a total of three times with FRT cells of different passages. The summarized data are shown in a scatter-dot plot, with a horizontal bar indicating the mean. Statistical comparisons were analyzed with one-way ANOVA followed by Sidak's multiple comparisons test (**, p < 0.01; ***, p < 0.001).

Article Snippet: The recovered surface proteins and 5% of the total lysate were separated by SDS-PAGE and blotted for the α subunit with HA-HRP antibodies (0.05 μg/ml, 3F10, Sigma), the γ subunit (0.3 μg/ml, StressMarq), PON3 (0.03 μg/ml, Sigma), or GAPDH (0.3 μg/ml, Proteintech, HRP-60004), as described previously ( 29 , 106 ).

Techniques: Expressing, Transfection, Plasmid Preparation, Labeling

Journal: Oncotarget

Article Title: Assessment of folate receptor-β expression in human neoplastic tissues

doi:

Figure Lengend Snippet: IHC was performed on a BioMax multi-cancer tissue microarray using the FR-β specific monoclonal antibody m909. The relative staining intensity, graded on a scale of 0 to 3, of positively staining cells within the tumor and stroma were determined. A ) Statistically significant differences ( p -value <0.05) using ANOVA or a t-test and correlations using a Spearman correlation ( p -value <0.05) between the average FR-β staining intensity and various pathological data are summarized. B ) Graphs for select FR-β staining intensity and various pathological data (error bars represent SEM) are shown. Lymph node involvement was based upon the AJCC/UICC stage - TxNxMx. Additional graphs and data can be found in the Supporting Information.

Article Snippet: The cancer tissue microarrays that were examined in this study were: i) a hepatocellular carcinoma tissue microarray from Emory University (Atlanta, GA), ii) a thymoma tissue microarray from Indiana University-Purdue University Indianapolis (Indianapolis, IN), iii) a custom multi-tumor tissue microarray (TMA-00300) from Asterand (Detroit, MI), iv) a high density multiple organ tumor and normal tissue microarray (MC5003) from US Biomax (Rockville, MD), and v) a breast tumor microarray (ARY-HH0056), a brain glioma tumor microarray (ARY-HH0138) and an endometrial carcinoma progression tumor microarray (ARY-HH0211) from Folio Biosciences (Columbus, OH).

Techniques: Microarray, Staining

Journal: Oncotarget

Article Title: Assessment of folate receptor-β expression in human neoplastic tissues

doi:

Figure Lengend Snippet: IHC was performed on a Asterand tissue microarray using the FR-β specific monoclonal antibody m909. The approximate percentage of positively staining cells within the tumor and stroma were determined. A ) Statistically significant differences ( p -value <0.05) using ANOVA or a t-test and correlations ( p -value <0.05) using a Spearman correlation between the percentage of FR-β staining cells and various pathological data are summarized. B ) Graphs for select FR-β staining and various pathological data (error bars represent SEM) are shown. Lymph node involvement was based upon the AJCC/UICC stage - TxNxMx. Additional graphs and data can be found in the Supporting Information.

Article Snippet: The cancer tissue microarrays that were examined in this study were: i) a hepatocellular carcinoma tissue microarray from Emory University (Atlanta, GA), ii) a thymoma tissue microarray from Indiana University-Purdue University Indianapolis (Indianapolis, IN), iii) a custom multi-tumor tissue microarray (TMA-00300) from Asterand (Detroit, MI), iv) a high density multiple organ tumor and normal tissue microarray (MC5003) from US Biomax (Rockville, MD), and v) a breast tumor microarray (ARY-HH0056), a brain glioma tumor microarray (ARY-HH0138) and an endometrial carcinoma progression tumor microarray (ARY-HH0211) from Folio Biosciences (Columbus, OH).

Techniques: Microarray, Staining