Ab216631

Total proteins from transfected AGS and MKN45 cells were isolated by RIPA buffer (R0010, Solarbio, Beijing, China) and quantified with the BCA protein quantification kit (ab102536, Abcam, Cambridge, UK) in line with the operating instructions. 20 μg protein samples were dissolved and electrically transferred onto a PVDF membrane (IPVH00010, EMD Millipore, Billerica, MA, USA). After blocking with 5% skim milk (D8340, Solarbio) at room temperature for 1 h, the membranes were incubated with primary antibodies against diverse proteins (RAD54B, 1:1000, ab168463; β-catenin, 1:500, ab16051; Axin, 1:1000, ab32197; cmyc, 1:20000, ab152146; MMP-7, 1:1000, ab216631; GAPDH, 1:2500, ab9485; all from Abcam) at 4°C overnight. Subsequently, the membranes were incubated with corresponding secondary antibodies for 2 h at room temperature and visualized by an ECL assay (P0018S, Beyotime, Shanghai, China). The band intensity was determined by ImageJ software (National Institutes of Health, USA).

Tumor tissues were fixed in 4% formaldehyde, dehydrated with gradient concentrations ethanol, embedded into parrafin (YA0011, Solarbio) and cut into sections with a thick of 5 μm. Sections were retrieved in 10 mM sodium citrate buffer (pH 6.0, P0083, Beyotime) for 15 min at 94°C. Following cooling to room temperature, sections were blocked with 1% bovine serum albumin (BSA, ST2249, Beyotime) for 30 min and then incubated with primary antibodies against RAD54B (1:200, ab238579, Abcam), Ki-67, c-myc (1:1000, ab32072, Abcam) and MMP-7 (1:500, ab216631, Abcam) respectively. Subsequently, sections were incubated with biotinylation-labeled secondary antibody (1:1000, ab207996, Abcam), re-stained with hematoxylin, and captured under a light microscope (Olympus). The relative level of RAD54B, Ki-67, cmyc and MMP-7 was determined as the ratio of the number of positive cells to the total number of cells.

Total proteins from A549 cells and tumor tissues were isolated using RIPA buffer (Beyotime) on ice. Then equal amount of protein samples were separated by 10–12% SDS-PAGE and transferred to PVDF membranes (Millipore), and then blocked with 5% bovine serum albumin (BSA; Beyotime). After being incubated with primary antibodies at 4°C overnight, samples were subjected to goat anti-rabbit HRP-conjugated secondary antibody (ab6721, 1:10,000, Abcam) at room temperature for 2 h. The bands were finally visualized by an ECL kit (Thermo Scientific), and then intensity was quantified by Image J 1.51 software (National Institutes of Health). Primary antibodies used included: RAB26 (ab198202; 1:000; Abcam), matrix metalloproteinases (MMP)2 (ab92536; 1:1000; Abcam), MMP7 (ab216631; 1:1000; Abcam), Bcl-2 (ab182858; 1:2000; Abcam), Bax (ab32503; 1:5000; Abcam), cleaved caspase 3 (#9661; 1:1000; Cell signaling pathway), caspase 3 (#9662; 1:1000; Cell signaling pathway), Bim (ab32158; 1:1000; Abcam), APAF-1 (ab234436; 1:1000; Abcam), Ki67 (ab16667; 1:1000; Abcam), proliferating cell nuclear antigen (PCNA; ab92552; 1:1000; Abcam), SMAD3 (ab208182; 1:1000; Abcam) and GAPDH (ab181602; 1:5000; Abcam).