Anti yap1 antibody

Protein was extracted from GAC cell lines and paired primary tissues using RIPA lysis buffer with proteinase inhibitor. Protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, CA) and 20 μg of protein mixed with 2 × SDS loading buffer was loaded per lane, separated by 12% SDS-polyacrylamide gel electrophoresis. Horseradish peroxidase (HRP) substrate solution was used for signal detection (Millipore, Billerica, MA). YAP1 protein was detected with a monoclonal anti-YAP1 antibody (1:10000 dilution, ab52771, Abcam, Cambridge, MA). Other primary antibodies were obtained from Cell Signaling Technology (Danvers, MA), CCND3 (1:2000, #2936), CCNE1 (1:1000, #4129), CDK6 (1:2000, #3136), p21 (1:1000, #2946), p27 (1:1000, #2552), p-Rb(Ser807/811) (1:1000, #9308), cleaved PARP(Asp214) (1:1000, #9541), BCL2(1:1000, #2870). The secondary antibodies were anti-Mouse IgG-HRP (1:30000 dilution, 00049039, Dako, Glostrup, Denmark) and anti-Rabbit IgG-HRP (1:10000, 00028856, Dako). The Western blot bands were quantified by ImageJ.

Immunohistochemistry staining was performed to assess the expression of YAP1 and CD74 in tumor tissue. The specimens were dewaxed and rehydrated routinely and then soaked in 0.3% H₂O₂ in methanol for 30 minutes at 37°C. The sections were then incubated with anti‐YAP1 antibody (1:100; Abcam) and anti‐CD74 antibody (1:100; Abcam) overnight at 4°C. Lastly, the sections were incubated with a secondary antibody (1:500; Dako) for one hour at room temperature. Semiquantitative results were obtained, as described previously. Basically, IHC images were captured with an FSX100 microscope (Olympus), and the German semiquantitative scoring method was employed to evaluate the scores. All stained sections, including nuclei, cytoplasms, and membranes, were evaluated and scored independently by two qualified pathologists with no prior knowledge of the clinicopathological outcomes of the patients. The intensity of YAP1 staining was scored as below to quantitatively group expression levels: 0 (no staining), 1 (weak staining, faint yellow), 2 (moderate staining, light brown), and 3 (strong staining, brown). Scores >2 were regarded as high expression. Multiple simultaneous evaluations were conducted to resolve the discrepancies (<5%).