Anti clu antibody

Immunohistochemistry was performed as described previously,²⁴ with some modifications. The antigen retrieval was performed by autoclaving in 0.01 M citrate buffer (pH 6.0) for 10 min at 120°C. CLU expression was detected by incubation with anti‐CLU antibody (Santa Cruz, #sc‐166907) and biotinylated goat anti‐mouse IgG (Nichirei, Tokyo, Japan). Evaluation of CLU expression was performed by two independent pathologists (NH and TU) based on the following criteria. The staining intensity in normal islet cells was used as an internal control for CLU expression in each section. Compared to the internal control, the predominant populations of tumor cells showing equivalent or more intensity were judged as positive (++), and those with less intensity as positive (+). Tumor cells without any CLU signal intensity were judged as negative (−).

The lysates from cultured cells or cryosections of frozen tissues were prepared and subject to SDS‐PAGE as described previously.²² The primary antibodies used in this study were anti‐CLU antibody (Santa Cruz, #sc‐5289, Dallas, TX, USA), anti‐phosphorylated ERK antibody (Thr²⁰²/Tyr²⁰⁴, Cell Signaling Technology, #4370), anti‐ERK antibody (Cell Signaling Technology, #9102), and anti‐GAPDH antibody (Santa Cruz, #sc‐32,233). Detection was performed with ECL Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ, USA). The signal intensity of CLU or GAPDH in each cell line was quantified using ImageJ. Then, the relative expression of CLU was calculated by dividing the quantified value of CLU by that of GAPDH. CLU was considered to show positive expression when the relative expression was >0.01. In addition, induced expression was considered to have occurred when the fold‐induction, calculated by dividing the relative expression of CLU after the PD0325901 treatment by that before the treatment, was >2.