Omni ecl enhanced pico light chemiluminescence kit

Neurons were lysed at 30 days with radioimmunoprecipitation assay (RIPA) lysis (Solarbio, Cat. #R0010, China), PMSF (Solarbio, Cat. # P0100, China), and phosphatase inhibitor (Solarbio, China) in proportions of 100:1:1, respectively. A BCA Protein Assay Kit (KeyGEN, Cat. #KGP902, China) was used for protein quantification. The proteins were used with 4–20% sodium dodecyl sulfate–polyacrylamide gel for electrophoresis and were transferred into the PVDF membrane (Merck Millipore, Cat. #ISEQ00010, Germany). They were then blocked with 5% nonfat milk for 90 min and incubated overnight with the primary antibody at 4°C. The following day, they were incubated with the secondary antibody. After that, an Omni-ECL™ Enhanced Pico Light Chemiluminescence Kit (EpiZyme, Cat. #SQ101, China) was added to capture the protein with a ChemiDoc™ MP Image System (Bio-RAD, Universal Hood III). The information about the antibodies is as follows: Kv4.3 (1:200; Affinity Biosciences); activating transcription factor 4 (ATF4) (1:200; Cell Signaling Technology); and C/EBP homologous protein (Bushart et al., 2018 (link)) (1:200, Proteintech). Goat anti-mouse IgG (H+L) -HRP (1:10,000; Bioworld) and goat anti-rabbit IgG (H+L) HRP (1:10,000; Bioworld) were used for Western blotting.

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.