Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Iron does not transcriptionally regulate ENaC alpha. The kidneys of 12-week-old WT or iron overloaded Hamp -/- mice were analyzed by real time PCR for gene expression of ENaC alpha. There was no significant difference in ENaC alpha gene expression of WT or spontaneously iron overloaded Hamp -/- mice under basal conditions ( A ). We confirmed that iron can regulate ENaC alpha gene transcription in mpkCCD cells, a cortical collecting duct cell line that is known to express ENaC alpha. 200 × 10 5 cells were serum-starved for 12 h and then cultured for 12 more hours in vehicle or FAC (100 uM). RNA was purified and then cDNA was synthesized. ENaC alpha gene expression was measured. Iron loading did not regulate ENaC alpha gene expression in mpkCCD cells ( B ), though it increased oxidative stress ( C ). One hundred uM FAC did not activate cell death program under the experimental conditions ( D ). Data from 2 independent experiments were pooled and analyzed. The statistical significance was determined by a 2-tailed Mann–Whitney test and is plotted as mean ± SEM. ** p < 0.001.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Purification, Synthesized, MANN-WHITNEY

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Immunohistochemistry analysis of ENaC alpha and MARCKS protein expression in Hamp -/- (KO) mice or wild-type (WT) mice. ( A ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in WT control mice. ( B ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in iron overloaded Hamp -/- mice. Data is representative of 3 mice in each group.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Immunohistochemistry, Expressing

Journal: American Journal of Physiology - Cell Physiology

Article Title: Effect of luminal flow on doming of mpkCCD cells in a 3D perfusable kidney cortical collecting duct model

doi: 10.1152/ajpcell.00405.2019

Figure Lengend Snippet: Primary antibodies used for immunofluorescence

Article Snippet: Bitplane, Concord, MA). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 1° Antibody Source Anti-acetylated α-tubulin (Lys40) rabbit monoclonal IgG (1:800) Cell Signaling (no. 5335) Anti-AQP2 rabbit polyclonal IgG (1:50) Alomone Laboratories (AQP-002) Anti-BKα chicken polyclonal IgG (1:100) Satlin Laboratory ( 101 ) (Aves Laboratories) Anti-ENaCα rabbit polyclonal IgG (1:50) StressMarq Biosciences (SPC-403) Anti-ENaCβ rabbit polyclonal IgG (1:50) StressMarq Biosciences (SPC-404) Anti-Na + /K + -ATPase rabbit monoclonal IgG (1:50) Abcam (ab76020) Open in a separate window AQP2, aquaporin 2; ENaC, epithelial Na + channel.

Techniques:

Journal: PLoS ONE

Article Title: Reduced Expression of Enac in Placenta Tissues of Patients with Severe Preeclampsia Is Related to Compromised Trophoblastic Cell Migration and Invasion during Pregnancy

doi: 10.1371/journal.pone.0072153

Figure Lengend Snippet: ( A ) real time-PCR result of the ENaC subunits expression in first term villous (left pannel), decidue tissues (middle pannel) and full term placenta (right pannel). α-ENaC is more abundenly expressed on the first term villous and decidual cells; while β-ENaC is more abundenly expressed on the full term villous. ( B ) The immunohistochemistry staining of α-ENaC in first term villous (a,c) and third term placenta of normal pregnancy(b,d). ( a ) The positive staining of α-ENaC is on the apical plasma membrane of the syncytiotrophoblast. Villous cytotrophoblastic cells are also stained. ( b ) Immunostainingof α-ENaC from the third term placenta. Very weak staining was seen in the syncytiotrophoblast cells. (c, d ) negative control with normal IgG in first term and third term placenta tissues. Magnification (X400). Scare Bar = 40 µM. ( C ) Western blot shows that HTR-8 cell express low level of β-ENaC, although high level of β-ENaC expression could be detected on full term placenta. ( D ) The effects of progesterone, estrogen on the α-ENaC expression in HTR-8/SVneo cells. Left pannel: HTR-8/SVneo cells were treated with progesterone (5 µM), estrogen (1 µM) respectively for 24 hr, and real time PCR was used to check α-ENaC expression. Relative expression levels between different groups were compared. The result shows that progesterone and estrogen could inhibited α-ENaC expression. Data were analyzed with one way ANOVA (*means p<0.05; ***means p<0.001). Right pannel: Western blot further confirmed that progesterone (5 µM) could reduce α-ENaC expression.

Article Snippet: After serum blocking, primary antibodies were added at 4°C overnight. β-ENaC rabbit anti-human polyclonal antibody (l∶200, abcam company, UK) and α-ENaC (1∶100, Cat No:10924-2-AP, Proteintech, China), γ-ENaC (1∶100, Cat No:13943-1-AP, Proteintech, China), were used as first antibodies for IHC staining, respectively.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry, Staining, Clinical Proteomics, Membrane, Negative Control, Western Blot

Journal: PLoS ONE

Article Title: Reduced Expression of Enac in Placenta Tissues of Patients with Severe Preeclampsia Is Related to Compromised Trophoblastic Cell Migration and Invasion during Pregnancy

doi: 10.1371/journal.pone.0072153

Figure Lengend Snippet: (A) Western blot result shows that α-ENaC RNAi can dose-dependently reduce the expression of ENaC. (B) The representative pictures in cell wounding and migration assay. The pictures shows the migration of HTR8/SVneo cells in the martrigel surface after 32 hr of scrape in different groups. The dot line shows the initial start point where the cell began the migration process. The cell number between in dot lines were count. The right is the statistics of the cell migration ability. The data are expressed as means ± SEM, of the three measurements (*means p<0.05).

Article Snippet: After serum blocking, primary antibodies were added at 4°C overnight. β-ENaC rabbit anti-human polyclonal antibody (l∶200, abcam company, UK) and α-ENaC (1∶100, Cat No:10924-2-AP, Proteintech, China), γ-ENaC (1∶100, Cat No:13943-1-AP, Proteintech, China), were used as first antibodies for IHC staining, respectively.

Techniques: Western Blot, Expressing, Migration

Journal: PLoS ONE

Article Title: Reduced Expression of Enac in Placenta Tissues of Patients with Severe Preeclampsia Is Related to Compromised Trophoblastic Cell Migration and Invasion during Pregnancy

doi: 10.1371/journal.pone.0072153

Figure Lengend Snippet: The immunohistochemistry staining of β-ENaC in placenta tissue of full-term placenta of normal pregnancy(A-C). The signal was detected in placental villous trophoblast cells, especially in syncytiotrophoblast (arrow, A) with less staining in cytotrophoblast and the staining was seen in extravillous cytotrophoblastic cells (arrowhead, B). (D, E) are staining of β-ENaC in placenta tissue from the preeclampsia patients. Reduced expression was noticed in placenta of preeclampsia patients (N = 3). (F) is negative control of the IHC staining. Magnification: X400, scare bar = 20 µM.

Article Snippet: After serum blocking, primary antibodies were added at 4°C overnight. β-ENaC rabbit anti-human polyclonal antibody (l∶200, abcam company, UK) and α-ENaC (1∶100, Cat No:10924-2-AP, Proteintech, China), γ-ENaC (1∶100, Cat No:13943-1-AP, Proteintech, China), were used as first antibodies for IHC staining, respectively.

Techniques: Immunohistochemistry, Staining, Expressing, Negative Control

Journal: PLoS ONE

Article Title: Reduced Expression of Enac in Placenta Tissues of Patients with Severe Preeclampsia Is Related to Compromised Trophoblastic Cell Migration and Invasion during Pregnancy

doi: 10.1371/journal.pone.0072153

Figure Lengend Snippet: β-ENaC expression in placenta of preeclampsia was reduced in RNA and protein level. (A) right bar chart represent the statistics of the real time PCR result, while left figure is the representative gel result of the RT-PCR result of the placenta samples. 12P,14P,17P were patients numbers of severe preeclampsia while left were normal controls. (B) Representative result of the Western blot result; The right is the statistic result (*means p<0.05).

Article Snippet: After serum blocking, primary antibodies were added at 4°C overnight. β-ENaC rabbit anti-human polyclonal antibody (l∶200, abcam company, UK) and α-ENaC (1∶100, Cat No:10924-2-AP, Proteintech, China), γ-ENaC (1∶100, Cat No:13943-1-AP, Proteintech, China), were used as first antibodies for IHC staining, respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: KCC3a, a Strong Candidate Pathway for K + Loss in Alkalemia

doi: 10.3389/fcell.2022.931326

Figure Lengend Snippet: Antibodies.

Article Snippet: ENaC (γ) , Rabbit polyclonal , 1:50 (IF) , StressMarq , SPC-405D.

Techniques:

Journal: Frontiers in Physiology

Article Title: Antiproteinuric and Hyperkalemic Mechanisms Activated by Dual Versus Single Blockade of the RAS in Renovascular Hypertensive Rats

doi: 10.3389/fphys.2021.656460

Figure Lengend Snippet: Effect of dual versus single RAS blockade on the expression of total and cleaved ENaC subunits in the non-clipped kidney of renovascular hypertensive rats. Samples isolated from the non-clipped kidney of renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) containing equal amounts of membrane proteins (75 μg for α-ENaC, β-ENaC, and γ-ENaC and 20 μg for actin) were subjected to SDS-PAGE, after which the proteins were transferred to a PVDF membrane and incubated with primary antibodies. Actin was used as a loading control. Representative immunoblots of electrophoresed renal membrane proteins and graphical representation of the relative expression of (A) Full-length (FL) α-ENaC, (B) cleaved α-ENaC, (C) β-ENaC, (D) cleaved γ-ENaC. The values represent individual measurements and the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs. 2K; ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. 2K-1C; &&& P < 0.001 and &&&& P < 0.0001 vs. 2K-1C + E; $$ P < 0.01 and $$$$ P < 0.0001 vs. 2K-1C + L.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% non-fat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and the following specific primary antibodies overnight (4°C): polyclonal antibody against podocin (1:1,000; Santa Cruz Biotech, sc-21009), polyclonal antibody against nephrin (1:1,000; Abcam, Cambridge, MA, Ab 58968), polyclonal antibody against megalin (1:50,000; a gift from Dr. Daniel Biemesderfer) , polyclonal antibody against cubilin (1:1,000; Santa Cruz Biotech, sc-20609), polyclonal antibody against the ClC-5, H + /Cl – exchange transporter 5 (1:1,000; Alpha Diagnostic International, CLC51-A, San Antonio, TX, United States) , polyclonal antibody against α-ENaC (1:1,000; Alomone Labs, ASC-030, Jerusalem, Israel), polyclonal antibody against β-ENaC (1:1,000; StressMarq Biosciences, SPC-404, Victoria, British Columbia, Canada), polyclonal antibody against cleaved γ-ENaC (1:1,000; Alomone Labs, ASC-011), or actin (1:5,000; Abcam ab179467).

Techniques: Expressing, Isolation, Control, Membrane, SDS Page, Incubation, Western Blot