30μg of proteins were separated by SDS-PAGE on 10% acrylamide gels, and proteins were electrically transferred to PolyScreen PVDF hybridization transfer membranes (Perkin Elmer, Boston, MA). Membranes were incubated overnight at 4°C with primary rabbit antibody for Scnn1a (1:500), Scnn1b and Scnn1g (1:1000) [32 (link)], CAP2/Tmprss4 [33 (link)] (1:200) and β-actin (1:1000, Sigma-Aldrich) and for 1 hour with donkey anti-rabbit IgG HRP-conjugated secondary antibody (1:10000, Amersham, Burkinghampshire, UK) (all antibodies in TBS-Tween 1% and dried milk 2%). The signal was revealed using SuperSignal West Dura detection system (Pierce, Rockford, IL) and quantified using ImageStudioTM Lite program (LI-COR). Kidney extracts from inducible renal tubule-specific Scnn1a KO mice, generated by interbreeding of Scnn1alox/lox mice [34 (link)] and Pax8::rtTA/LC1 mice [35 (link)], were used as control for Scnn1a-specific signals on Western blot (control non-doxycycline-induced animals [Ctrl WT], control doxycycline-induced animals [Ctrl KO]). The same strategy was applied for Scnn1b- and Scnn1g-specific bands [36 ]. The specificity of the primary antibody for CAP2/Tmprss4 has been described previously and extensively tested in vitro using the Xenopus oocyte expression system [33 (link)], and corroborated using protein extracts from CAP2/Tmprss4 knock-out mice that were used as control.
Image studio lite program
Manufactured by LI COR
Sourced in United States
Image Studio Lite is a software program designed for image acquisition and analysis. It provides core functions for capturing, processing, and managing digital images from various imaging devices. The software offers essential tools for image editing, measurement, and data presentation without subjective interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Donkey anti rabbit igg hrp conjugated secondary antibody, by Cytiva (2 mentions)
β actin, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyscreen pvdf hybridization transfer membranes, by PerkinElmer (1 mentions)
Amersham hybond ecl, by GE Healthcare (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Immobilon hrp substrate, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bm chemiluminescence western blotting kit, by Roche (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Odyssey near infrared imager, by LI COR (1 mentions)
Irdye800 conjugated anti rabbit igg, by Rockland Immunochemicals (1 mentions)
Western lightning plus ecl substrate, by PerkinElmer (1 mentions)
Semi dry transfer apparatus, by Bio-Rad (1 mentions)
6 protocols using image studio lite program
1
Western Blot Analysis of Sodium Channel Proteins
2
Western Blotting Analysis of Renal Proteins
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Kidneys were homogenized as described26 (link). 30 µg of proteins (120 µg for HKA2) were separated by SDS-PAGE on 10% acrylamide gels, and proteins were electrically transferred to nitrocellulose membranes (Amersham Hybond-ECL, GE Healthcare) and incubated overnight at 4 °C with primary antibody against HKA2 (1:200, a gift from Dr. Crambert, INSERM, Paris, France), NKA (1:10000)75 (link), NCC (1:500)76 (link), pT53-NCC (1:1000)76 (link), AQP2 (1:1000, Santa Cruz, Dallas, TX, USA), NKCC2 (1:2000)77 (link), pT96/T101-NKCC2 (1:200)27 (link), GR (1:1000, Santa Cruz, Dallas, TX, USA), pS211-GR (1:200, Bioss, Boston, MA, USA), PR (1:1000, Santa Cruz, Dallas, TX, USA), AR (1:1000, Santa Cruz, Dallas, TX, USA), ERα (1:200, Santa Cruz, Dallas, TX, USA), MR (1:50)75 (link), Scnn1a (1:500)78 (link), Scnn1b (1:1000)78 (link), Scnn1g (1:1000)78 (link), β-actin (1:1000, Sigma-Aldrich) and α tubulin (1:1000, Santa Cruz, Dallas, TX, USA), and for 1 hour with donkey anti-rabbit IgG HRP-conjugated secondary antibody (1:10000, Amersham, Bukinghampshire, UK) or donkey anti-mouse IgG HRP-conjugated (Jackson Immuno Research, Baltimore, PA, USA) (all antibodies in TBS-Tween 1% and dried milk 2%). The signal was revealed using SuperSignal West Dura detection system (Pierce, Rockford, IL) and quantified using ImageStudioTM Lite program (LI-COR).
3
Western Blot Analysis of Denatured Proteins
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Cells were lysed in a non-reducing loading buffer containing 63 mM Tris-HCl pH 6.8, 10% glycerol, and 2% sodium dodecyl sulfate (SDS). Protein concentrations were quantified using the Pierce BCA protein assay kit (Thermo-Scientific). Proteins (40 μg) were resolved under denaturing conditions by 7.5–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (EMD Millipore). Membranes were blocked in 5% non-fat milk in TBS-T buffer (25 mM Tris-HCl, pH 7.4, 125 mM NaCl, and 0.1% Tween 20) for 1 hour, washed 3 times for 5 minutes with TBS-T buffer, and incubated with appropriate primary antibodies at 4 °C overnight. Membranes were rinsed 3 times for 5 minutes with TBST-T buffer; membranes were then incubated with appropriate HRP-conjugated secondary antibodies (Jackson Immunoresearch), diluted in TBS-T for 1 hour at room temperature. Immune complexes were detected by enhanced chemiluminescence (Pierce ECL, Thermo-Scientific; Immobilon HRP Substrate, Millipore) and quantified with the Image Studio Lite program (Li-COR Biosciences). Antibodies used in this study are listed in the Supplementary Table 1 . Full length unprocessed blot images are provided in the Supplementary Materials.
4
Western Blot Analysis of Metabolic Regulators
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Aliquots (30 μg) of total protein extracted from cell lysates and liver tissue were loaded onto a 10% SDS-PAGE machine under reducing conditions, and were then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA), which were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 1 h. Subsequently, membranes were immunoblotted with specific antibodies (p-AMPKα Thr172, 1:500; LKB1, AMPKα 1/2, SIRT1, PPARα and β-actin) overnight at 4 °C. Afterward, membranes were incubated with a peroxidase-conjugated secondary antibody (1:16,000) for 1 h at room temperature. Bands were detected using the BM Chemiluminescence Western Blotting Kit (Roche Applied Science, Penzberg, BY, Germany), and visualized through ChemiDoc XRS+ (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometry analysis was performed using the ImageStudio Lite program (LI-COR Biosciences, Lincoln, NE, USA). All band-density quantifications were normalized with β-actin.
5
Quantitative Analysis of UGDH Oligomerization
6
Immunoblotting Procedure for Protein Analysis
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Lysates for immunoblots were prepared by addition of 2X Laemmli buffer. After boiling for 5 min, proteins were resolved on 10% bis-tris acrylamide gels by SDS-PAGE, and transferred to PVDF membranes using a semi-dry transfer apparatus (Bio-Rad). Membranes were blocked with either 5% nonfat milk in 1X TBST or protein-free TBS blocking buffer (Pierce). Primary antibody incubation occurred overnight at 4°C with gentle agitation. Multiple washes were performed with 1X TBST. Secondary antibodies were HRP-conjugated. Chemiluminescence was activated using Western Lightning Plus ECL Substrate (PerkinElmer). All immunoblots were quantified via the Image Studio Lite program (LI-COR Biosciences) and normalized to a loading control.
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