Cep192

RIPA buffer (Beyotime, China) was used to extract the protein following the appropriate steps. BCA Protein Assay Kit (Beyotime, China) was used to measure the concentration of extracted protein.Cells were collected and lysed with RIPA buffer. Proteins were quanti ed using a bicinchoninic acid assay (Thermo Scienti c), resolved by SDS-PAGE, and transferred onto PVDF membranes (Millipore, Billerica, USA). Antibody detection was conducted using an enhanced chemiluminescent substrate kit (Yeasen).
Antibodies against CEP192 (Bioss, 1:1000, China), E-cadherin (Abcam, 1:500, Cambridgeshire, UK), KRT-8 (Abcam, 1:1000, Cambridgeshire, UK) were used to the related protein level. β-actin (1:1000, Abcam, UK) and GAPDH (1:2500, Abcam, UK) were used for normalization.

IHC was performed using 4-µm-thick sections of representative formalin-xed tissue blocks. Brie y, the slides were dewaxed in xylene, passed through graded alcohols, and placed into 0.01 mol/L phosphatebuffered saline (PBS; pH = 7.4). The slides were then pretreated with 1.0 mM citrate, pH 6.0 (Invitrogen), in a steam pressure cooker for epitope retrieval and were washed in PBS. Next, they were incubated with 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity and were subsequently incubated with a monoclonal rabbit anti-human CCL18 antibody (Biodragon, 1:125, China), CEP192(BIOSS 1: 250, China) at 4 °C overnight. On the following day, the slides were washed with PBS and incubated with an anti-rabbit secondary antibody (Dako) for 60 min at room temperature. After being washed in PBS, the slides were stained with DAB+ (Dako) and then counterstained for 1 min with Harris hematoxylin (BASO), differentiated in 1% hydrochloric acid in alcohol, dehydrated, and mounted. All PT and LNM specimens were stained using the same protocol.