Irdye 800 conjugated goat anti rabbit igg second antibodies

Western blot was carried out as we previously described (Song et al., 2019 (link)). Briefly, total proteins were extracted from HeLa cells with SPINT1-AS1 or SPINT-AS1-del stable overexpression and HeLa cells with SPINT1-AS1 stable silencing using RIPA lysis buffer (Beyotime, Shanghai, China). Total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by being transferred to polyvinylidene difluoride membrane (Millipore). After block using 5% non-fat milk at room temperature for 2 h, the membranes were incubated with primary antibodies against β-catenin (1:1,000, #8480, Cell Signaling Technology) or GAPDH (1:1,000, #97166, Cell Signaling Technology) overnight at 4°C. After further incubation with IRDye 700-conjugated goat anti-mouse IgG or IRDye 800-conjugated goat anti-rabbit IgG second antibodies (Invitrogen), the membranes were scanned on the Odyssey infrared scanner (Li-Cor, Lincoln, NE, United States). GAPDH was used as endogenous control for to quantitate β-catenin protein levels.

Total cell lysates were extracted from transfected cells 48 hours after transfection or stable cell lines with RIPA lysis buffer (Beyotime, Shanghai, China) added with protease inhibitors (Beyotime) in accordance with the instructions. Equal quantities of proteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were incubated with 5% non‐fat milk for 90 minutes at room temperature. Next, the membranes were incubated with primary antibodies against EZH2 (Cell Signaling Technology, Danvers, MA, USA) or GAPDH (Cell Signaling Technology) overnight at 4°C. The membranes were further incubated with IRdye 700‐conjugated goat anti‐mouse IgG or IRdye 800‐conjugated goat anti‐rabbit IgG second antibodies (Invitrogen) and detected on an Odyssey infrared scanner (Li‐Cor, Lincoln, NE, USA). GAPDH was employed as an endogenous control for the quantification of EZH2 protein expression.