Nuclear plasma separation kit

Total cellular protein was extracted from cell lines or tumor tissues with radio-immunoprecipitation assay lysis buffer (P0013C) purchased from Beyotime Biotechnology (Shanghai, China). Nuclear proteins were separated with Nuclear Plasma Separation Kit (P0028, Beyotime Biotechnology). The concentration of protein was quantified using a BCA Kit (P0012, Beyotime Biotechnology). The cell lysate was then mixed with 5× loading buffer (P0015, Beyotime Biotechnology) and separated using SDS-PAGE with gels ranging from 8% to 12%. After transferring the proteins onto poly(vinylidene fluoride) membranes (Millipore, Danvers, MA, United States) and blocking with 5% BSA at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with secondary antibodies for 1 h and visualized in Tanon 5200 Chemiluminescent Imaging System (Shanghai, China) using an ECL Detection Kit (Millipore). Relative protein expression was normalized with Lamin B1 for the detection of NF-κB p65 or β-actin for the detection of the whole protein lysate and analyzed with Image J 1.52a (NIH, Bethesda, MD, United States).

Western blotting was performed as described previously 18 (link). Protein samples were collected, concentrated, and SDS-PAGE protein loading buffer was added. Samples were analyzed using SDS-PAGE and then transferred to a PVDF membrane. The membrane was immediately placed in the prepared western washing solution and rinsed for 1-2 min. The membrane was then treated with blocking solution, followed by incubation with the first antibody overnight. The next day it was incubated with the second antibody (1:2500) at room temperature for 2 h. The primary antibodies used were: anti-ATF4 (sc-22800, Santa Cruz, USA, 1:200), anti-β-catenin (sc-393501, anti-Santa Cruz, USA, 1:200), anti-cyclin D1 (sc-20044, Santa Cruz, 1:200), anti-MMP7 (sc-515703, Santa Cruz, 1:200), anti-Histone H3(sc-517576, Santa Cruz, 1:500) and anti-GADPH (sc-59540, Santa Cruz, USA, 1:1000). The membrane was treated with enhanced chemiluminescence (ECL) for protein bands, and the signals were detected using a BioImaging System. To determine the relative protein expression in nucleus and cytoplasm separately, nuclear and cytoplasmic protein samples were prepared by using the Nuclear plasma separation Kit (Beyotime, China).