Monoclonal rabbit anti phospho akt ser473 antibody

A Western blotting analysis was performed as described previously [24 (link)]. The primary antibodies were: polyclonal rabbit anti-FOXM1 antibody (Santa Cruz Biotechnology, CA, USA, 1:100), monoclonal rabbit anti-phospho-AKT (ser473) antibody (Cell Signaling Technologies, Tokyo, Japan, 1:500), monoclonal rabbit anti-AKT (pan) antibody (Cell Signaling Technologies, Tokyo, Japan, 1:500), monoclonal rabbit anti-phospho-MEK antibody (Cell Signaling Technologies, Tokyo, Japan, 1:1000) monoclonal rabbit anti-MEK antibody (Cell Signaling Technologies, Tokyo, Japan, 1:1000). β-actin was used as a loading control.

Immunohistochemical analyses were performed as described previously [9 (link)]. Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan). An isotype monoclonal mouse antibody (clone MG2a-53; abcam, Tokyo, Japan) and an isotype monoclonal rabbit antibody (Cell Signaling Technologies, Tokyo, Japan) were used as negative controls. The slides were mounted using aqueous medium and viewed under a microscope. The intensity of staining was classified as (-) (the same or weaker than the adjacent epidermis) or (+) (stronger than the adjacent epidermis). The samples were divided into two groups (positive or negative for FOXM1) according to the results of immunostaining, and the positive rate of FOXM1 was determined. Stained sections were scored according to the percentage of stained melanoma cells: 75–100%, 50–74%, 25–49%, 1–24% or negative. The samples were evaluated independently by two observers (A.M. and S.F.) in a blinded manner.