Western blot analysis of Phospho-mTOR (S2448) on different lysates with Mouse anti-Phospho-mTOR (S2448) antibody (HA600094) at 1/1,000 dilution. Lane 1: NIH/3T3 cell lysate treated with serum-starvation overnight Lane 2: NIH/3T3 cell lysate treated with Insulin (100nM) for 10 minutes after serum-starvation overnight Lysates/proteins at 10 µg/Lane. Predicted band size: 288 kDa Observed band size: 255 kDa Exposure time: 2 minutes; 10% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA for 1 hour at room temperature. The primary antibody (HA600094) at 1/1,000 dilution was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunohistochemical analysis of paraffin-embedded human liver tissue with Mouse anti-Phospho-mTOR (S2448) antibody (HA600094) at 1/100 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA600094) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.