AKT Signaling Pathway Antibodies

Immunohistochemistry analysis of FGFR1 (pYpY653/654) showing staining in the cytoplasm and membrane of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H₂O₂-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-FGFR1 (Tyr53, Tyr654) Polyclonal Antibody (Cat. No. 44-1140G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Western blot analysis was performed on whole cell extracts (30 µg lysate) of MCF7 (Lane 1) and Hep G2 (Lane 2). The blot was probed with FOXO3 Polyclonal Antibody (Cat. No. PA5-27145, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 0.25 µg/mL, 1:4,000 dilution). A 71 kDa band corresponding to FOXO3A was detected across the cell lines tested.

Flow cytometry analysis of Phospho-4EBP1 pThr37 in Jurkat cells using a Phospho-4EBP1 (Thr37) Recombinant Rabbit Monoclonal Antibody (52H37L2) (Cat. No. 700238) at a dilution of 0.1 µg. Cells were fixed and permeabilized using FIX and PERM (Cat. No. GAS-004) reagent, and detection was performed using an Alexa Fluor 488 goat anti-rabbit IgG (black) compared to a control without primary antibody (gray). Pre-incubation with the phosphopeptide decreased the signal (blue) whereas incubation with the non-phosphopeptide did not (red).