Phospho pdk1

The 4^th mammary glands excised from mice were snap frozen in liquid nitrogen, then lysed in cold RIPA buffer (1 × PBS, 1% NP-40, 0.5% sodium dexoycholate, 0.1% SDS, protease inhibitor cocktail tablet [Roche] and 1 mM PMSF [Beyotime Biotechnology]). Lysates were incubated on ice for 20 minutes with frequent vortexing and cleared twice by centrifugation (13,200 rpm,10 minutes, 4 °C). Protein was subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). Membranes were blocked for 60 minutes at room temperature in 5% non-fat milk/Tris-buffered saline/0.1% Tween (TBST). After being washed with TBST, the blots were incubated in primary antibodies for 3 h, which were PS6 (1:2000, CST), S6 (1:2000, CST), TSC1 (1:1000, Proteintech), PDK1(1:1000, ABclonal), phospho-PDK1 (1:1000, Bioworld) phospho-AKT308 (1:1000, Bioworld), phospho-AKT473 (1:1000, CST), AKT1 (1:1000, CST) and β-actin (1:1000, Bioworld). Antibody detection was performed according to the manufacturer’s instructions with ECL Plus Western Blotting Detection System (Genstar, Beijing, China) and developed on film.

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.