Western Blot Analysis of IRS and PI3K Signaling

The expression of proteins was determined by western blot as previously described (18 (link)). The concentration of protein was detected using a BCA kit (Beyotime, Shanghai, China). Then, 30 μg of total protein was loaded onto 8% SDS polyacrylamide gels and transferred to the polyvinylidene fluoride membrane. The membrane then was blocked by 5% BSA (Sigma) for 1 h and incubated with primary antibodies at 4°C overnight. The next day, the membrane was washed with PBS for three times and then followed by incubation with HRP-labeled goat anti-rabbit IgG (Abcam, America) secondary antibody at 37°C for 1 h. Signals were detected by using the ECL detection kit (Merck Millipore) and normalized to the expression of GAPDH. The antibodies included Total insulin receptor substrate (IRS)-1 (ab52167, 1:1,000, Abcam), phosphorylated IRS-1 (p-IRS-1) at PY896 (sc-560, 1:500, Santa Cruz), total IRS-2 (ab52606, 1:1,000, Abcam), phosphorylated IRS-2 (p-IRS-2) at S731 (sc-1555-R,1:500, Santa Cruz), total PI3K (ab154598,1:1,000, Abcam), phosphorylated PI3K (p-PI3K) at P85 (ab191606, 1:1,000, Abcam), phosphorylated Akt (p-Akt) at Ser-473 (ab192623, 1:1,000, Abcam), GAPDH (ab9485, 1:1,000, Abcam). The absorbance of each band was quantitated using the Image Pro Plus Software.

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Liu C., Zhou X.R., Ye M.Y., Xu X.Q., Zhang Y.W., Liu H, & Huang X.Z. (2021). RBP4 Is Associated With Insulin Resistance in Hyperuricemia-Induced Rats and Patients With Hyperuricemia. Frontiers in Endocrinology, 12, 653819.

Publication 2021

BCA kit HRP-labeled goat anti-rabbit IgG ECL detection kit ab52167 sc-560 ab154598 ab191606 ab192623 ab9485

Anti igg Antibodies Antibody Gapdh Goat Insulin receptor substrate 1 Insulin receptor substrate 2 Membrane Pi3k Polyacrylamide gels Polyvinylidene fluoride Protein Rabbit Sc 560 Western blot

Corresponding Organization :

Other organizations : Central South University, Second Xiangya Hospital of Central South University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression of proteins determined by western blot
Concentration of protein detected using a BCA kit
Signals detected by using the ECL detection kit and normalized to the expression of GAPDH

control variables

30 μg of total protein loaded onto 8% SDS polyacrylamide gels
Membrane blocked by 5% BSA for 1 h
Primary antibodies incubated at 4°C overnight
HRP-labeled goat anti-rabbit IgG secondary antibody incubated at 37°C for 1 h
GAPDH expression used for normalization

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

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