Sab2900314

Cells were lysed in RIPA lysis buffer and proteins were isolated. The target antibodies and 150 µl incubation buffer were added to the protein lysate and rotated at 4 °C overnight. Then 80 µl suspended protein A + G beads (Protein A/G Agarose Beads, Proteintech) were added (centrifuged 3 times with PBS) and incubated at 4 °C for 4 h. After centrifugation at 4 °C for 30 s, the supernatant was discarded, and samples were subjected to western blotting. For the ubiquitination experiment, HCT116 cells were treated with 20 µM MG132 (an inhibitor of ubiquitination proteasome) for 6 h. The ubiquitination levels of ABI1 were detected by immunoprecipitation and western blotting.
The antibodies used were as follows: CBLC antibody (1:1000, SAB2900314, Sigma); ABI1 antibody (1:500, 66,609–1-Ig, Proteintech) and ubiquitin antibody (1: 5000, 10,201–2-AP, Proteintech).

Tissues were embedded in paraffin, sectioned at 5-µm thick sections, and stained with hematoxylin and eosin (H&E) for routine histological examination. For IHC staining, sections were deparaffinized, hydrated, and subjected to heat-induced antigen retrieval. Endogenous peroxidase was inactivated using 3% H₂O₂ at room temperature for 15 min. Then sections were blocked with 1% BSA for 15 min and incubated with primary antibodies (CBLC, 1:200, SAB2900314, Sigma, Germany or Ki-67, 1:200, ab15580, Abcam, UK) overnight at 4 °C, followed by incubation with the goat anti-rabbit IgG-HRP secondary antibody (1:500, 31,460, ThermoFisher, USA) at 37 °C for 60 min. The antigen sites were detected by DAB. Then sections were stained with hematoxylin for 3 min, and images were photographed under a microscope.

Total protein was extracted using RIPA buffer (Solarbio). Quantification was performed with the BCA protein concentration assay kit (Solarbio). Approximately 20 µg total protein was passed through 10% SDS polyacrylamide gels, and then transferred to PVDF membranes (Millipore, USA). The membranes were incubated with the primary antibodies (CBLC, 1:1000, SAB2900314, Sigma; ERK, 1:500, 13–6200, Invitrogen; p-ERK, 1:1000, A18196, Abclonal; ABI1, 1:10,000, 66,609–1-Ig, Proteintech; CDK1, 1:1000, A22347, ABclonal; cyclin B, 1:2000, 55,004–1-AP, Proteintech; E-cadherin, 1:1000, A20798, ABclonal and Vimentin, 1:1000, A19607, ABclonal) at 4 °C overnight, followed by incubation with the corresponding secondary antibodies (1:3000, goat anti-rabbit IgG-HRP, SE134, Solarbio; 1:3000, or goat anti-mouse IgG-HRP, SE131, Solarbio) in the dark at room temperature for 60 min. The blots were developed with enhanced chemiluminescence (ECL) reagent (Solarbio), and data analysis was performed by Gel-Pro-Analyzer software.