Ab72593

Immunohistochemistry was performed to detect Ki-67 and DUSP4 expression in the mouse tumor tissues. Briefly, tumor tissues were fixed in 10% formalin for paraffin block preparation. tumor slices were permeabilized with blocking buffer (5% BSA/0.25% TX-100 in PBS) and incubated with the Ki-67 (1:100, ab15580, Abcam) or DUSP4 (1:50, ab72593, Abcam) antibody overnight at 4 ^oC. After washing with PBS, samples were incubated with HRP-conjugated secondary antibody (Invitrogen, Carlsbad, CA) before analysis by microscopy (PerkinElmer, Waltham, MA). Ki67- and DUSP4-positive cells were quantified using Image J software.

The cells subjected to different treatments were harvested in radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China), and the protein concentration was measured using the bicinchoninic acid protein assay (Thermo Fisher Scientific). Proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, blocked with 5% skimmed milk in Tris-buffered saline, and reacted with primary antibodies against E-cadherin (1:3000; ab1416, Abcam, Cambridge, MA, USA), vimentin (1:5000; #5741, CST, Danvers, MA, USA), DUSP4 (1:2000; ab72593, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2000; ab9485, Abcam). GAPDH was used as an internal control. Thereafter, the membranes were washed with TBST and further incubated with a horseradish peroxidase-conjugated secondary antibody (#7074, #7076, CST) at a 1:2000 dilution for 2 h at room temperature. The bands were visualized using ECL solution (Pierce).

Sections (4 μm thick) were obtained from each TMA block, and we performed deparaffinization and rehydration with routine techniques (e.g., immersing in xylene and graded ethanol). Then, antigen retrieval (heat-induced, 100 °C for 20 min in sodium citrate buffer) and blocking of endogenous peroxidase activity (for 15 min in S2023 peroxidase-blocking solution, Dako, Glostrup, Denmark) were performed. For IHC staining of DUSP4, rabbit anti-DUSP4 polyclonal antibody (1:200, ab72593, Abcam, Cambridge, MA, USA) was used. Detection was achieved using an EnVision Detection System (K5007, Dako, Glostrup, Denmark). A series of IHC staining procedures were performed according to the manufacturer’s instructions using a Bond Max Automated Immunostainer (Leica Biosystems, Nussloch, Germany). DUSP4 expression was defined as nuclear staining of tumor cells. We classified each case as DUSP4 negative (lack of staining or staining in <10%) or DUSP4 positive (staining in ≥10%). There is no standard cutoff for DUSP4 expression in RCC. However, it is common to set a cutoff of 10% for novel biomarkers [17 (link),18 (link)]. The staining intensity was not assessed in this study due to concerns of subjectivity and reproducibility. Two pathologists (Bang, S. and Paik, S.S.) evaluated DUSP4 expression without consideration of clinicopathological data.