Journal: Physiological Reports

Article Title: Resistance to doxorubicin‐induced proteinuria and proteolytic activation of ENaC in 129S2 / SvPas mice

doi: 10.14814/phy2.70667

Figure Lengend Snippet: Doxorubicin injection in 129S2/SvPasCrl males does not enhance proteolytic cleavage of ENaC's γ subunit. (a) A schematic is shown depicting cleavage sites in ENaC's γ subunit and the antibody used for immunoblot. (b) Immunoblot of ENaC's γ subunit from deglycosylated whole kidney lysates. Each lane represents a kidney lysate from one mouse. (c) Quantification of full‐length γ subunit or cleavage products (normalized to stain‐free gel protein intensity). Full‐length γ subunit and furin‐cleaved C‐terminal fragment abundances increase in doxorubicin‐treated mice compared to untreated controls. Distally cleaved γ subunit protein abundance does not increase significantly. (d) Abundance of the furin‐cleaved or distally cleaved proteolytic fragments relative to the total (full‐length + furin‐cleaved + distally cleaved) γ subunit abundance is not enhanced by treatment with doxorubicin. (Ctrl, control mice untreated with doxorubicin; Doxo, doxorubicin‐treated mice. Numbers in pair‐wise comparisons represent p values, as determined using Student's t ‐test.)

Article Snippet: Lysates were then subjected to SDS‐PAGE and immunoblot using an antibody directed against the ENaC γ subunit's C‐terminus (StressMarq, #SPC‐405) as previously described (Ray et al., ).

Techniques: Injection, Western Blot, Staining, Quantitative Proteomics, Control

Journal: Frontiers in Medicine

Article Title: Case Report: Functional investigation of the γENaC G532S mutation presenting as mild PHA-1B3

doi: 10.3389/fmed.2025.1605057

Figure Lengend Snippet: The γENaC-G532S mutation decreases the ENaC sodium current. (A) Current traces of αβγENaC-WT (top) and αβγENaC-G532S (bottom) are shown during exposure to the solutions indicated by the horizontal bars, at a holding potential of −80 mV. The left and right representative traces result from the protocol measuring amiloride-sensitive current; middle traces show currents measured during exposure to 5 μg/mL trypsin. The current amplitude scale is the same for the 6 traces, as indicated. (B) Amiloride-sensitive currents (I Na ) measured in oocytes expressing αβγENaC-WT ( n = 31) or αβγENaC-G532S ( n = 57), measured before, during, and after treatment with trypsin. *** p < 0.001, Kruskal–Wallis’s one-way ANOVA test, followed by the Dunn’s multiple comparison test. (C) Normalized amiloride-sensitive currents. The current amplitudes recorded from a given batch of oocytes were normalized to the average ENaC-WT amplitude of that batch for this analysis. (D) Fold increase in I Na measured after treatment with trypsin: in each oocyte, the amiloride-sensitive current amplitude after trypsin exposure was normalized to that measured before trypsin. (E) Amiloride-sensitive currents (I Na ), peak and plateau, measured during treatment with trypsin. *** p < 0.001, Kruskal–Wallis’s one-way ANOVA test, followed by the Dunn’s multiple comparison test. (F) I peak /I plateau of amiloride-sensitive current during trypsin treatment was calculated in each oocyte by normalizing the transient peak amplitude to the corresponding plateau current. (C,D,F) Mann–Whitney’s U -test was performed for statistical analysis. (B–F) Bars represent the mean ± SEM. Note that all data of this figure are from oocytes injected with RNA encoding the three subunits α, β and γ, αβγENaC-WT or αβγENaC-G532S.

Article Snippet: ENaC γ subunit , Rabbit , StressMarq , SPC-405D , 1 k.

Techniques: Mutagenesis, Expressing, Comparison, Injection

Journal: Frontiers in Medicine

Article Title: Case Report: Functional investigation of the γENaC G532S mutation presenting as mild PHA-1B3

doi: 10.3389/fmed.2025.1605057

Figure Lengend Snippet: Biochemical analysis of WT and mutant ENaC. (A) Representative Western blot analysis of the total (=membrane-enriched) and the cell surface (biotinylated) fractions expression of each of the ENaC subunits, in oocytes injected with either αβγWT or αβγG532S cRNAs. Bands corresponding to the full-length (FL) or cleaved (Cl) forms of α- and γENaC are indicated. Western blots of the endogenous Na + /K + -ATPase α subunit were performed in parallel for normalization of protein recovery. (B-C) Densitometric quantification of Western blots from three independent experiments. The band intensities are normalized to the corresponding Na + /K + -ATPase band intensities to correct for differences in protein recovery. The values thus obtained for each experiment were normalized with that of the corresponding WT subunit. (B) Band intensities of each of the ENaC subunits in membrane-enriched fractions. (C) ENaC subunit expression from biotinylated fractions.

Article Snippet: ENaC γ subunit , Rabbit , StressMarq , SPC-405D , 1 k.

Techniques: Mutagenesis, Western Blot, Membrane, Expressing, Injection

Journal: Frontiers in Medicine

Article Title: Case Report: Functional investigation of the γENaC G532S mutation presenting as mild PHA-1B3

doi: 10.3389/fmed.2025.1605057

Figure Lengend Snippet: The γENaC-G532S mutation decreases the ENaC sodium current. (A) Current traces of αβγENaC-WT (top) and αβγENaC-G532S (bottom) are shown during exposure to the solutions indicated by the horizontal bars, at a holding potential of −80 mV. The left and right representative traces result from the protocol measuring amiloride-sensitive current; middle traces show currents measured during exposure to 5 μg/mL trypsin. The current amplitude scale is the same for the 6 traces, as indicated. (B) Amiloride-sensitive currents (I Na ) measured in oocytes expressing αβγENaC-WT ( n = 31) or αβγENaC-G532S ( n = 57), measured before, during, and after treatment with trypsin. *** p < 0.001, Kruskal–Wallis’s one-way ANOVA test, followed by the Dunn’s multiple comparison test. (C) Normalized amiloride-sensitive currents. The current amplitudes recorded from a given batch of oocytes were normalized to the average ENaC-WT amplitude of that batch for this analysis. (D) Fold increase in I Na measured after treatment with trypsin: in each oocyte, the amiloride-sensitive current amplitude after trypsin exposure was normalized to that measured before trypsin. (E) Amiloride-sensitive currents (I Na ), peak and plateau, measured during treatment with trypsin. *** p < 0.001, Kruskal–Wallis’s one-way ANOVA test, followed by the Dunn’s multiple comparison test. (F) I peak /I plateau of amiloride-sensitive current during trypsin treatment was calculated in each oocyte by normalizing the transient peak amplitude to the corresponding plateau current. (C,D,F) Mann–Whitney’s U -test was performed for statistical analysis. (B–F) Bars represent the mean ± SEM. Note that all data of this figure are from oocytes injected with RNA encoding the three subunits α, β and γ, αβγENaC-WT or αβγENaC-G532S.

Article Snippet: ENaC α subunit , Rabbit , StressMarq , SPC-403S , 1 k.

Techniques: Mutagenesis, Expressing, Comparison, Injection

Journal: Frontiers in Medicine

Article Title: Case Report: Functional investigation of the γENaC G532S mutation presenting as mild PHA-1B3

doi: 10.3389/fmed.2025.1605057

Figure Lengend Snippet: Biochemical analysis of WT and mutant ENaC. (A) Representative Western blot analysis of the total (=membrane-enriched) and the cell surface (biotinylated) fractions expression of each of the ENaC subunits, in oocytes injected with either αβγWT or αβγG532S cRNAs. Bands corresponding to the full-length (FL) or cleaved (Cl) forms of α- and γENaC are indicated. Western blots of the endogenous Na + /K + -ATPase α subunit were performed in parallel for normalization of protein recovery. (B-C) Densitometric quantification of Western blots from three independent experiments. The band intensities are normalized to the corresponding Na + /K + -ATPase band intensities to correct for differences in protein recovery. The values thus obtained for each experiment were normalized with that of the corresponding WT subunit. (B) Band intensities of each of the ENaC subunits in membrane-enriched fractions. (C) ENaC subunit expression from biotinylated fractions.

Article Snippet: ENaC α subunit , Rabbit , StressMarq , SPC-403S , 1 k.

Techniques: Mutagenesis, Western Blot, Membrane, Expressing, Injection