Western Blot Analysis of Metabolic Regulators

Total lysates containing 30 μg protein for liver, skeletal muscle and vWAT were loaded in each lane and were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membrane and membranes were blocked with 5% (w/v) nonfat dry milk (in TBS-T). Primary antibodies were diluted in TBS with 0.01% (v/v) Tween 20 (TBS-T) and 5% (w/v) bovine serum albumin (BSA), while secondary antibodies were diluted in TBS with 0.01% (v/v) Tween 20 (TBS-T) and 5% (w/v) nonfat dry milk. Membranes were probed with the following antibodies: polyclonal anti PTP1B (Abcam- 1:750 dilution), monoclonal anti-Total OXPHOS (Abcam- 1:500 dilution), polyclonal anti SIRT-1 (Abcam- 1:1,000 dilution), polyclonal anti P-AMPKα (Thr172) (Cell Signaling- 1:1,000 dilution), polyclonal anti AMPKα (Cell Signaling- 1:1,000 dilution), monoclonal anti IRS1 (Cell Signaling- 1:500 dilution), monoclonal anti P-IRS1 (Tyr612) (Invitrogen- 1:1,000 dilution), monoclonal anti P-IRS1 (Ser307) (Cell Signaling- 1:1,000 dilution), monoclonal anti FAS (Santa Cruz Biotechnology- 1:1,000 dilution), monoclonal anti SREBP1c (Santa Cruz Biotechnology- 1:1,000 dilution), monoclonal anti SPOT14 (Santa Cruz Biotechnology- 1:1,000 dilution), polyclonal anti PGC1α (Millipore- 1:1,000 dilution), monoclonal anti Mfn2 (Abcam- 1:1,000 dilution), monoclonal anti DRP1 (Cell Signaling- 1:1,000 dilution), monoclonal anti Catalase (Merck- 1:750 dilution), monoclonal anti SOD-2 (Santa Cruz Biotechnology- 1:500 dilution), polyclonal anti GPX1 (GeneTex- 1:1,000 dilution), monoclonal anti GPX4 (Abcam- 1:1,000 dilution), polyclonal anti PRDX-3 (Abcam- 1:1,000 dilution) and monoclonal anti B-ACTIN antibody (Bioss Antibodies- 1:1,000 dilution). As secondary antibodies we used peroxidase anti rabbit IgG (Vector Laboratories- 1:2,000–1:4,000–1:10,000 dilution) and peroxidase anti-mouse IgG (Vector Laboratories- 1:4,000 dilution). Horseradish peroxidase-conjugated secondary antibodies were used for signal detection by enhanced chemiluminescence using the Chemi Doc system and related software (Hercules, CA, United States of America).

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Petito G., Giacco A., Cioffi F., Mazzoli A., Magnacca N., Iossa S., Goglia F., Senese R, & Lanni A. (2023). Short-term fructose feeding alters tissue metabolic pathways by modulating microRNAs expression both in young and adult rats. Frontiers in Cell and Developmental Biology, 11, 1101844.

Publication 2023

Actin Anti igg Antibodies Bovine serum albumin Catalase Chemiluminescence Dilution Gels Gpx4 Horseradish peroxidase Irs1 Liver Membranes Mfn2 Milk Monoclonal antibody Mouse Nitrocellulose Peroxidase Pgc1α Protein Ptp1b Rabbit Sds page Signal detection Sirt Skeletal muscle Spot14 Srebp1c Tween 20 Vector

Corresponding Organization : University of Campania "Luigi Vanvitelli"

Other organizations : University of Sannio, University of Naples Federico II

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Variable analysis

independent variables

Total lysates containing 30 μg protein for liver, skeletal muscle and vWAT were loaded in each lane

dependent variables

Protein expression levels of PTP1B, Total OXPHOS, SIRT-1, P-AMPKα (Thr172), AMPKα, IRS1, P-IRS1 (Tyr612), P-IRS1 (Ser307), FAS, SREBP1c, SPOT14, PGC1α, Mfn2, DRP1, Catalase, SOD-2, GPX1, GPX4, and PRDX-3

control variables

Protein loading was kept constant at 30 μg per lane
Blocking was performed using 5% (w/v) nonfat dry milk (in TBS-T)
Primary antibodies were diluted in TBS with 0.01% (v/v) Tween 20 (TBS-T) and 5% (w/v) bovine serum albumin (BSA)
Secondary antibodies were diluted in TBS with 0.01% (v/v) Tween 20 (TBS-T) and 5% (w/v) nonfat dry milk

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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