Hpa014711

Immunohistochemical staining was performed on representative tissue sections cut from formalin-fixed, paraffin-embedded tissues at 3-μm thickness as our previous study [24 (link)] with a few modifications. Slides were deparaffinized with xylene, rehydrated with ethanol, heated by microwave for retrieval of antigen epitopes in a 10 mM citrate buffer (pH 6) for 7 min. Endogenous peroxidase was quenched by 3% H₂O₂. Slides were washed with Tris buffered saline for 15 min and then incubated with a primary monoclonal antibody against EMP2 (1:20; HPA014711, Sigma-Aldrich), CREB1 (1:40, sc-186, Santa Cruz) and pCREB1(S133) (1:50, sc-7978, Santa Cruz), for 1 h, followed by antibody detection using a ChemMate EnVision™ kit (K5001; DAKO, Glostrup). Two pathologists (CF Li and HY Huang) blinded to clinicopathological information and patient outcomes, independently interpreted the immunostainings. The immunointensity was scored based on the extent of moderately to strongly-stained tumor cells exhibiting combined membranous and cytosolic (EMP2) or nuclear [CREB and pCREB(S133)] staining, and labeled as 0+, < 5%; 1+, ≥ 5%, but < 25%; 2+, ≥ 25%, but < 50%; 3+, ≥ 50%, but < 75%; and 4+, ≥ 75%, respectively. A specimen showing EMP2 staining less than 1+ was regarded as loss of EMP2 expression. For CREB1 and pCREB1(S133), 4+ staining were regarded as high expression.

Cell lysates were prepared with RadioImmuno Precipitation Assay buffer (Upstate). Lysates containing equal amounts of protein were separated by SDS-PAGE and electroblotted onto Immobilon™-P Transfer Membrane (Millipore). The filters were individually probed with specific primary antibody. Protein bands were detected by the Western Lightning Chemiluminescence Reagent Plus Kit (Perkin-Elmer Life Sciences) with horseradish peroxide labeled secondary antibody as suggested by the manufacturer and visualized on a VersaDoc Image System (Bio-Rad). The intensity of bands was quantified by densitometry and normalized to ACTB in each lane. Anti-human EMP2 (1:100, HPA014711, Sigma-Aldrich), anti-cyclin dependent kinase 1 (CDK1; 1:200, sc-54, Santa Cruz), anti-pCDK1(Y15) (1:250, BD Transduction Laboratories™), anti-WEE1 (1:1000, #4936, Cell Signaling), anti-CCNB1 (1:500, sc-245, Santa Cruz), anti-pCDC25C(S216) (1:1000, #4901, Cell Signaling) and anti-pCREB1(S133) (1:200, sc-7978, Santa Cruz) were used as primary antibodies for immunoblotting analysis and anti-ACTB (1:3000, Chemicon) was served as a loading control.