Goat polyclonal anti mest antibody

The cells were lysed in buffer containing 50 mM Tris-HCl (pH 6.9; Sigma), 2% sodium dodecylsulfate (SDS; Nacalai), 6% 2-mercaptoethanol (Sigma), and 10% glycerol. Protein samples (20 μg) were subjected to 4%–20% SDS-polyacrylamide gel electrophoresis and subsequently transferred onto an Immuno-Blot PVDF membrane (Bio-Rad). The membrane was treated with a mouse monoclonal anti-β-actin antibody (Santa Cruz Biotechnology) at a dilution of 1 : 1000 or goat polyclonal anti-MEST antibody (Abcam) at a dilution of 1 : 200, followed by biotinylated anti-mouse IgG (Nichirei Biosciences, Inc., Tokyo, Japan) or anti-goat IgG (Nichirei) as appropriate. After the membrane was washed thoroughly, the reactive bands were visualized using the ECL Select western blotting detection system (GE healthcare, Buckinghamshire, UK) and Image Quant LAS 500 (GE).

Cells were fixed with 4% PFA and 0.5% dimethyl sulfoxide (Wako) in PBS for 20 min. After blocking with 2% BSA in PBS for 1 h, the cells were incubated for 1 h with a goat polyclonal anti-MEST antibody (Abcam) at a dilution of 1 : 200 or mouse monoclonal anti-Ki67 antibody (Invitrogen) at a dilution of 1 : 100 and then washed with PBS. The cells were then incubated for 1 h with an Alexa Fluor 488-conjugated chicken anti-goat antibody (Invitrogen) at a dilution of 1 : 200. The cells were washed with PBS and then counterstained with DAPI (Vector Laboratories, Burlingame, CA). The cytoskeleton was stained with Phalloidin-Tetramethylrhodamine B isothiocyanate (Sigma). Cells were imaged and analyzed under the Biozero digital microscope.