Irs 1

Western blotting was carried out as previously described5 (link) using rabbit antibodies against the following proteins: IR (1:800), IRS-1 (1:800), p-AKT (1:800), GSK-3β (S9) (all 1:800), or glyceraldehyde phosphate dehydrogenase (GAPDH) (1:10,000) (all from Bioworld, San Diego, CA, USA); GAPDH was used as an internal reference standard. Secondary antibodies were peroxidase-conjugated goat anti-rabbit IgG (H + L) (ZSGB-BIO, Beijing, China).

Formalin-fixed paraffin-embedded sections were placed in pressure cooker (full pressure for 3 min) with appropriate 0.01 M sodium citrate (pH 6.0) for antigen retrieval. Subsequently, sections were incubated in blocking solution consisting of 5% BSA and 10% (v/v) normal goat serum in PBS at room temperature for 1 hour. The primary antibodies TSC1((1:100,abclonal), phosphor-S6 (1:100, CST), phospho-PDK1 (1:100, Bioworld), phospho-AKT308 (1:100, Bioworld), phospho-AKT473 (1:100, CST), IRS-1 (1:100, Bioworld), ERα (1:30, Proteintech), cyclin D1 (1:50, Abclonal), cyclin E (1:100, Immunoway), CDK4 (1:100, Abclonal), CDK2 (1:100, Proteintech), p-Rb (1:100, Abclonal) and p27kip1 (1:100, Abclonal) were then applied and incubated at 4 °C overnight. Appropriate secondary antibodies were applied for 1 hour. Nuclei were counterstained with hematoxylin.