Column tissue cell protein extraction kit

The RIPA lysis buffer (Beyotime, China) containing a proteinase and phosphatase inhibitor cocktail (Bimake, USA) was used to isolate the total protein. Proteins were extracted from the tissues using a Column Tissue&Cell Protein Extraction Kit (Epizyme, China). The protein concentration was measured with the Enhanced BCA Protein Assay Kit (Beyotime, China). Aliquots of protein were subsequently separated on 10%–12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% skim milk, the membranes were treated with primary antibodies and the corresponding secondary antibodies. Finally, protein bands were detected utilizing enhanced chemiluminescence (ECL, Millipore, USA) and visualized using an imaging system (Tanon, China). Primary antibodies were as follows: PICK1 (1:1,000, CST, 85325), GAPDH (1:3,000, CST, 5174), Flag (1:5,000, Sigma, F1804), β-catenin (1:10,000, Abcam, ab32572), c-Myc (1:1,000, CST, 5605), CyclinD1 (1:10,000, Abcam, ab134175), Actin (1:1,000, CST, 4970), Lamin B1 (1:10,000, Proteintech, 66095-1-Ig), a-Tubulin (1:5,000, Sigma, T6074), His (1:1,000, CST, 12698), HA (1:5000, Sigma, H6908), Phospho-GSK-3β (Ser9) (1:1,000, CST, 5558).

WB assay was used to determine the changes of related proteins. In brief, we cultured SW480/SW620 cells into the 6-well plate and exposed them to TNF-α or CXCL10 stimulation for a specified time. Cells were collected and the Column Tissue&Cell Protein Extraction Kit (Epizyme) was employed to extract total protein. Protein concentration was measured by Omni-Easy™ Instant BCA Protein Assay Kit (Epizyme). The PAGE Gel Fast Preparation Kit (Epizyme) was utilized to prepare the gel for electrophoresis; proteins were then transferred to PVDF membranes, followed by blocking using 5% skim milk contained within TBST for a period of 1.5 h before overnight incubation using primary antibodies under 4 °C. Primary antibodies included including anti-N-cadherin, E-cadherin, fibronectin, CXCR3, Vimentin, p38/p-p38, p65/p-p65, PI3K/p-PI3K, Akt/p-Akt, GSK-3β/p-GSK-3β, Snail, Twist, RhoA, cdc42, Slug, ZEB-1, IKKα, IKKβ, IκBα, β-catenin, LaminB and GAPDH rabbit antibodies (1:1200, CST). Then, cells were incubated using HRP-labeled anti-rabbit IgG antibody (1:12000, CST) for 2 h, The Omni-ECL™Enhanced Pico Light Chemiluminescence Kit (Epizyme) was used for protein luminescence detection on a Bio-Rad gel imaging instrument.