Cell Lysate Preparation and Western Blot

Cell lysate preparation and western blot analysis were performed in adherence to established procedures [15 (link), 20 (link)]. Whole-cell lysates were obtained using a kit (Servicebio Co. Ltd., Wuhan, China). Nuclear and cytoplasmic fractions were obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN, Nanjing, China). The following primary antibodies were used: Akt (#4691, Cell Signaling), P-Akt (#4060, Cell Signaling), Nrf2 (ab89443, Abcam), TBP (ab51841, Abcam), SOD1 (10269-1-AP, Proteintech), SOD2 (24127-1-AP, Proteintech), CAT (21260-1-AP, Proteintech), GSH-Px1 (#PB0205, BosterBio), and β-actin (Servicebio Co. Ltd.). After incubation with horseradish peroxidase-conjugated secondary antibodies, specific bands were visualized by enhanced chemiluminescence using a Bio-Rad ChemiDoc XRS (Bio-Rad, USA) and quantified by Image Lab™ Software.

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Wu Q., Lu K., Zhao Z., Wang B., Liu H., Zhang S., Liao J., Zeng Y., Dong Q., Zhao N., Han B, & Du Y. (2019). Blockade of Transient Receptor Potential Vanilloid 4 Enhances Antioxidation after Myocardial Ischemia/Reperfusion. Oxidative Medicine and Cellular Longevity, 2019, 7283683.

Publication 2019

Nuclear and Cytoplasmic Protein Extraction Kit P-Akt ab89443 ab51841 SOD2 (24127-1-AP, CAT (21260-1-AP, β-actin ChemiDoc XRS

Actin Antibodies Cell Chemiluminescence Cytoplasmic Horseradish peroxidase Nrf2 Protein Sod2 Western blot analysis

Corresponding Organization : Xuzhou Central Hospital

Other organizations : South Central University for Nationalities

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Variable analysis

independent variables

Cell lysate preparation and western blot analysis methods

dependent variables

Protein expression levels of Akt, P-Akt, Nrf2, TBP, SOD1, SOD2, CAT, GSH-Px1, and β-actin

control variables

Established procedures for cell lysate preparation and western blot analysis [15, 20]
Whole-cell lysates obtained using a kit (Servicebio Co. Ltd., Wuhan, China)
Nuclear and cytoplasmic fractions obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN, Nanjing, China)
Horseradish peroxidase-conjugated secondary antibodies
Enhanced chemiluminescence detection using a Bio-Rad ChemiDoc XRS (Bio-Rad, USA) and Image Lab™ Software

controls

No positive or negative controls were explicitly mentioned in the information provided.

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