Journal: JCI Insight

Article Title: Metalloproteinase PAPP-A regulation of IGF-1 contributes to polycystic kidney disease pathogenesis

doi: 10.1172/jci.insight.135700

Figure Lengend Snippet: (A) Relative mRNA expression of IGF-1 pathway components in kidneys of 7.5-month-old C57BL/6J (n = 4–6) and Pkd1RC/RC mice (n = 5–7). PCR data are expressed relative to Gapdh. (B) Correlation between kidney size (total kidney weight relative to heart weight) and renal Pappa mRNA expression in Pkd1RC/RC mice (n = 15). (C) Pappa mRNA levels in various tissues of WT (n = 3–5) and Pkd1RC/RC mice (n = 4–6). (D) Pappa mRNA levels in WT (n = 6) and Pkd2WS25/– (n = 5) mouse kidneys (16 weeks old). (E) ELISA analysis of PAPP-A protein levels in human ADPKD cystic fluid (n = 6) compared with normal serum reference. (F) Immunolocalization of PAPP-A in normal and ADPKD human kidneys. (G) Western blot analysis of PAPP-A protein levels in normal human RCTE and ADPKD cystic epithelial cells (9-12); graph shows quantification relative to tubulin. Scale bars: 200 μm. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed (for Igf1, 1-tailed) Student’s t test.

Article Snippet: Human ADPKD kidney tissue slides were produced in-house under an institutionally approved IRB protocol, and normal human kidney tissue slides were purchased from Novus Biologicals.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Science advances

Article Title: Three-dimensional histological electrophoresis enables fast automatic distinguishment of cancer margins and lymph node metastases.

doi: 10.1126/sciadv.adg2690

Figure Lengend Snippet: Fig. 2. Verification of the 3D histological electrophoresis distinguishing tumors from adjacent nonmalignant tissues. (A) Fluorescence images demonstrated the differences between the IR-780–labeled proteins in 4T1 tumor, breast, and muscle lysates following the separation using the 2D SDS-PAGE. (B) Western blotting for detecting overexpressed albumin and AKT1 in the 4T1 tumor sample. (C) Fraction 1 was selected to distinguish tumor and muscle tissues. Quantification of the Fraction 1–to–Fraction 2 (actin) ratio as annotated in (A), albumin-to-actin ratio, and AKT1-to-actin ratio as annotated in (A) (**P < 0.05). (D) IHC staining of albumin and AKT1 levels in 4T1 tumor and muscle tissues. Scale bar, 50 μm. (E) Western blotting for detecting albumin and AKT1 levels in a set of 4T1 tumor samples collected after 3, 7, 21, and 35 days of inoculation, respectively. (F) Schematic representation of the workflow of the 3D histological electrophoresis for tissue sections. (G) Two samples with either muscle&muscle or 4T1 tumor&muscle combination were subjected to the 3D histological electrophoresis. (H) The tumor-to-muscle ratios were analyzed and plotted before/after the separation by 3D histological electrophoresis (n = 3 mice per group, ****P < 0.05). (I) By collecting a set of 4T1 tumor samples after 3, 7, 21, and 35 days of inoculation, the tumor-to-muscle ratios were analyzed and plotted after the 3D histological electrophoresis (n = 3 mice per group). (J) NIR images and signal quantifi- cation of the seven fractionated layers for the muscle&muscle sample after the 3D histological electrophoresis. Scale bar, 1 cm. (K) NIR images and signal quantification of seven fractionated layers for the tumor&muscle sample following the 3D histological electrophoresis. Layers 2, 3, and 4 were observed with the maximum signal differ- ence between tumors and muscles, primed for assessing the tumor-positive margins. Scale bar, 1 cm.

Article Snippet: AKT1 was purchased from MedChemExpress.

Techniques: Electrophoresis, Fluorescence, Labeling, SDS Page, Western Blot, Immunohistochemistry, Muscles

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