To determine if 17α-E2 altered hepatic insulin sensitivity, we evaluated phosphorylation status of AKT and FOXO1 following an insulin bolus. Liver was homogenized in RIPA Buffer (Cell Signaling, Danvers, MA) with protease and phosphatase inhibitors (Boston Bioproducts, Boston, MA). Total protein was quantified using BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). Proteins were separated on an Any kD Criterion TGX Stain-Free Protein Gel (Biorad, Hercules, CA) at 75V for 150 min in Running Buffer (Cell Signaling, Danvers, MA) and transferred to a 0.2 µm pore size nitrocellulose membrane, (Biorad, Hercules, CA) at 75V for 90 min on ice. Primary antibodies used were pS256 FOX01 (Abcam ab131339, 1:1000), FOX01a (Abcam ab52857, 1:1000), pS473 AKT (Abcam ab81283, 1:3000), pan-AKT (Abcam ab179463, 1:10000), GAPDH (Abcam ab9485, 1:2500). Primary antibody detection was performed with IRDye 800CW Infrared Rabbit (LI-COR Biotechnology, Lincoln, NE) at 1:15,000 concentration. GAPDH was diluted in 5% dry milk (Cell Signaling, Danvers, MA), all other antibodies were diluted in 5% BSA (Cell Signaling, Danvers, MA). Blot imaging was done on Odyssey Fc Imaging System (LI-COR Biotechnology, Lincoln, NE) with a two-minute exposure time at 800λ, and protein detection and quantification were performed using Image Studio Software (LI-COR Biotechnology, Lincoln, NE).
0.2 m pore size nitrocellulose membrane
Manufactured by Bio-Rad
Sourced in United States
The 0.2 µm pore size nitrocellulose membrane is a laboratory filtration product. It has a pore size of 0.2 micrometers, which allows for the effective filtration of small particles and molecules.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (3 mentions)
Running buffer, by Cell Signaling Technology (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Image studio software, by LI COR (2 mentions)
Sodium deoxycholate, by Merck Group (2 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Rabbit anti mouse igg peroxidase antibody, by Merck Group (2 mentions)
Goat anti rabbit igg h l peroxidase conjugated antibody, by Abcam (2 mentions)
Protease and phosphatase inhibitors, by Boston BioProducts (1 mentions)
Any kd criterion tgx stain free protein gel, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Trans blot turbo transfer pack, by Bio-Rad (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
C4731, by Merck Group (1 mentions)
Mab901, by R&D Systems (1 mentions)
8 protocols using 0.2 m pore size nitrocellulose membrane
1
Hepatic Insulin Sensitivity Assay
2
Western Blot Analysis of Cellular Proteins
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Sorted cells were lysed in RIPA Lysis and Extraction Buffer (Pierce) containing protease inhibitors (Pierce). Cell lysates were then sonicated with a 15-second pulse at a power output of 2 using the VirSonic 100 (SP Industries Company). Protein concentrations were next determined with a BCA protein assay (Pierce). Proteins were resolved with SDS-PAGE utilizing 4–15% Mini-PROTEAN TGX Precast Gels (Biorad) and transferred to Trans-Blot Turbo Transfer Packs (Biorad) with a 0.2-µm pore-size nitrocellulose membrane (Biorad). Nitrocellulose membranes were blocked in 5% nonfat milk in 0.05% (v/v) Tween 20 (PBST) (Corning). Membranes were then incubated with primary antibodies overnight. After washing in PBS containing 0.05% (v/v) Tween 20 (PBST), membranes were incubated with secondary antibodies (Thermo Scientific). After washing, membranes were developed with the Clarity Western ECL Blotting Substrate (Biorad). Histone H3 was used as a loading control for all blots. Please see Supplementary Figure 9 for uncropped scans of representative blots.
3
Immunoblotting Protein Analysis Workflow
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Protein samples were separated by 4–12% Bis–Tris Gels (Invitrogen) and transferred to a 0.2 µm pore-size nitrocellulose membrane (Bio-Rad). The primary antibodies used were: mouse monoclonal anti-human MMP1 (clone MAB901, 1:1000, R&D Systems, RRID:AB_2144271), goat polyclonal anti-human THBS1 (AF3074, 1:1000, R&D Systems, RRID:AB_2201958), rabbit polyclonal anti-TSG101 (A303-507A, 1:1,000, Bethyl Laboratories Inc., RRID:AB_10971167), rabbit polyclonal anti-CD63 (PA5-78995, 1:1000, Invitrogen, RRID:AB_2746111), mouse monoclonal anti-human ALIX (clone OTI1A4, VMA00273, 1:2000, Bio-Rad PrecisionAb western blot validated), sheep polyclonal anti-GM130/GOLGA2 (AF8199-SP, 1:1000, R&D Systems), rabbit anti-calnexin (C4731, 1:2000, Sigma-Aldrich, RRID:AB_476845). Pre-stained protein ladder was from Abcam (ab234592).
4
Protein Extraction and Western Blot Analysis
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Cell lysis and protein extraction were carried out using the RIPA buffer (1 M Tris-HCl pH 8 (PanReac AppliChem, ITW Reagents), 0.5 M EDTA (Thermo Fisher Scientific), Triton X-100 (Sigma-Aldrich), 10% sodium deoxycholate (Sigma-Aldrich), 10% SDS (Sigma-Aldrich) and 3 M NaCl (Thermo Fisher Scientific)), supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Further, 20 µg of protein from each sample were separated by SDS-PAGE and transferred to a 0.2 µm pore-size nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h with 5% BSA (PanReac AppliChem, ITW Reagents) in 1x TBS-T (0.1% Tween20, Bio-Rad) and incubated with specific antibodies HMGA2 (Proteintech, 20795-1-AP), HMGB1 (Abcam, ab18256), HMGB2 (Proteintech, 14597-1AP) and ɑ-tubulin (Sigma-Aldrich, T9026) overnight at 4 °C. Then, membranes were washed with 1x TBS-T and incubated with rabbit antimouse IgG (Sigma-Aldrich) or goat antirabbit IgG (Abcam, Cambridge, UK), both conjugated with peroxidase. HRP substrate (GE Healthcare, Life Sciences) was used for chemiluminescent detection, and image acquisition was performed using a Chemidoc Imaging System (Bio-Rad).
5
Cell Lysis and Protein Extraction Protocol
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For lysing cells and to extract protein RIPA buffer was used, adding a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). For the SDS-PAGE assay, a total of 20 µg/sample was loaded in the acrylamide gel. Proteins were transferred from the gel to 0.2 µm pore-size nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were then blocked for 1 h using 5% BSA (PanReac AppliChem, ITW Reagents) in 1X TBS-T (0.1% Tween20, Bio-Rad). After membrane blocking, the following antibodies were used for blotting proteins: anti-HMGA1 (ab129153; Abcam), anti-PARP1 (51-66396R; BD Bioscience, Franklin Lakes, NJ, USA), anti-cleaved Caspase 3 (#9661; Cell signaling, Danvers, MA, USA), anti-phospho-S6 (#4858; Cell signaling), anti-S6 (#2217; Cell signaling) and anti-α-tubulin (Sigma-Aldrich, T9026). Membranes were consequently incubated with Rabbit Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich) or Goat Anti-Rabbit IgG H&L peroxidase-conjugated antibody (Abcam) secondary antibodies. For chemiluminescent detection, the HRP substrate was used. Image acquiring was performed using the ChemiDoc Imaging System (Bio-Rad). ImageJ was used to quantify protein expression levels.
6
Western Blot Analysis of ISG15 Expression
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Cell lysis and protein extraction was carried out using the RIPA buffer [1 M Tris–HCl pH 8 (PanReac AppliChem, ITW Reagents), 0.5 M EDTA (Thermo Fisher Scientific), Triton™ X-100 (Sigma-Aldrich), 10% sodium deoxycholate (Sigma-Aldrich), 10% SDS (Sigma-Aldrich) and 3 M NaCl (Thermo Fisher Scientific)], supplemented with protease and phosphatase inhibition cocktails (Sigma-Aldrich). 20 µg protein sample were separated by SDS-PAGE, then transferred to 0.2 µm pore-size nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h using 5% BSA (PanReac AppliChem, ITW Reagents), in 1X TBS-T (0.1% Tween20, Bio-Rad). Subsequently, the following antibodies were incubated in the same buffer for 16 h at 4 ºC: ISG15 (Santa Cruz Biotechnology, sc-166755), α-tubulin (Sigma-Aldrich, T9026). Membranes were washed with 1X TBS-T, then incubated with Rabbit Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich) or Goat Anti-Rabbit IgG H&L peroxidase-conjugated antibody (Abcam, Cambridge, UK). HRP substrate was used for chemiluminescent detection and image acquisition was performed using a Chemidoc Imaging System (Bio-Rad). ISG15 expression was relativised against INT-SFT values.
7
Western Blot Analysis of SCD1 Protein
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Liver homogenates not used for the liver triglyceride analyses were spun at 17,000 rpm for 15 min at 4 °C and the supernatant was collected. Total protein was quantified using a Pierce BCA kit (ThermoFisher Scientific, Waltham, MA, USA). Proteins were separated on Any kD Criterion TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA) at 75 V for 150 min in running buffer (Cell Signaling). Protein was then transferred to 0.2 µm pore-size nitrocellulose membranes (Bio-Rad) at 75 V for 90 min on ice. Both primary antibodies utilized have been commercially validated and include SCD1 (Cell Signaling; 1:1000) and GAPDH (Abcam, Waltham, MA, USA; 1:2500). Primary antibody detection was performed with IRDye 800CW Infrared Rabbit antibody (LI-COR Biotechnology, Lincoln, NE, USA) at a concentration of 1:15,000. Imaging was done on an Odyssey Fc Imaging System (LI-COR Biotechnology) protein quantification was performed using Image Studio Software (LI-COR Biotechnology).
8
Western Blot Analysis of Hepatic Proteins
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Hepatocytes and cholangiocytes proteins were extracted in lysis buffer (50 mM Tris⋅HCl pH 7.5, 1 mM EGTA, 1 mM EDTA pH 8.0, 50 mM sodium fluoride, 1 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.27 M sucrose, 1% Triton X-100, 0.1 mM PMSF, 0.1% 2-mercaptoethanol, 1 µg/mL leupeptin, and 1 µg/mL aprotinin). Extracts were separated by SDS–PAGE and transferred to 0.2-µm pore-size nitrocellulose membranes (Bio-Rad). Blots were probed with primary antibodies to ErbB2 (Cat# ab16901, Abcam; RRID: AB_443537 ), phospho-ERK (Cat#9101, Cell Signaling Biotechnology; RRID: AB_330744 ), JNK1 (Cat# sc-1648, Santa Cruz Biotechnology; RRID:vAB_675868 ), FGF-15 (Cat# sc-514647, Santa Cruz Biotechnology), FGF-21 (Cat# RD281108100, BioVendor Laboratory Medicine; RRID: AB_2034054 ), and Vinculin (Cat#V4505, Sigma; RRID:AB_477617 ). All antibodies were used at 1:1,000 dilution. The membranes were washed and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (GE Healthcare), and immune complexes were detected using an enhanced chemiluminescent substrate (Clarity Western ECL substrate; Bio-Rad).
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