Rabbit anti zeb1

The LN18 and U87 cells were cultured in 6-well plates and treated by drugs. The cells were washed with 4°C PBS twice, collected and lysed in RIPA Buffer. Then the lysate was centrifuged for 15 minutes and the supernatants were retained. The protein extracts were separated on 8%–12% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk in 0.1% TBST for 1 hour and then incubated with primary antibodies against rabbit anti-N-cadherin, rabbit anti-Vimentin, rabbit anti-Snail, rabbit anti-Slug, rabbit anti-ZEB1, rabbit anti-p-AKT, rabbit anti-AKT, rabbit anti-p-mTOR, rabbit anti-mTOR, rabbit anti-MMP-9, rabbit anti-Bmi1, rabbit anti-Musashi1, rabbit anti-Sox2 (All of the above antibodies were procured from Cell Signaling Technology)overnight at 4°C, followed by incubation with secondary antibodies (1:5000 dilution, Boster, Wuhan, China). Equal lane loading was confirmed using a monoclonal antibody against β-tubulin (ORIGENE). After washing with the TBS-T buffer, the membranes were scanned with the Beijing Sage Creation Science Co., Ltd.

The following antibodies were used: mouse anti- FLAG M2 (Sigma-Aldrich), rabbit anti-HA tag (Abcam), rabbit anti-E-cadherin (Cell Signaling Technology), mouse anti-E-cadherin (DAKO), rabbit anti-ZEB1 (Cell Signaling Technology), rabbit anti-Snail1 (Cell Signaling Technology), rabbit anti-Twist1 (BIORAD), mouse anti-β-catenin (Santa Cruz Biotechnology), mouse anti-TCF4 (Santa Cruz Biotechnology), mouse anti-β-catenin (Sigma-Aldrich), rabbit anti-phospho-STAT3 (Abcam), rabbit anti-STAT3 (Abcam), rabbit anti-galectin-3 (Abcam), goat anti-Trop-2 (R&D), and HRP-conjugated mouse anti-FLAG M2 antibodies (Sigma-Aldrich). Rabbit anti-phospho-Trop-2 antibodies were prepared as described previously (23 (link)).

Cells were lysed in RIPA lysis buffer and the protein concentration was determined. Total proteins were fractionated using SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were blocked in 5% skim milk in TBST buffer containing 0.1% Tween 20 and then incubated with indicated primary antibodies (Rabbit anti-N-cadherin, 1: 1000, Millipore; Mouse anti-Vimentin, 1: 500, Millipore; Rabbit anti-E-cadherin, 1: 1000, Abgent; Rabbit Anti-ZEB1, 1: 1000, Cell Signaling) for 2 h at room temperature. HRP-conjugated secondary antibodies (HRP Goat anti-Rabbit IgG Antibody, 1: 5000, Abgent; HRP Goat anti-Mouse IgG Antibody, 1: 5000, Abgent) were incubated at room temperature (RT) for 1 h and detected using the enhanced chemiluminesence detection system.Total protein was extracted from untreated and the cells treated by transfecting miR-200a and subjected to western blot analysis as described to evaluate the expression of E-cadherin, N-cadherin, ZEB1 and vimentin. The data was adjusted against loading control using β-actin.

SW480 colorectal cancer cell line (ATCC® CCL-228) was maintained in RPMI640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in atmosphere containing 5% CO₂. To overexpress Klf4-GFP and GFP-control in SW480 cell line, cells were transiently transfected with 3 μg plasmid DNA (per well in a six-well plate) using Lipofectamine 2000 reagent (ThermoFisher Scientific) according to manufacturer’s instructions. The cell lysates were collected using Laemmli buffer and subjected to Western blot analysis with the following antibodies: rabbit anti-KLF4 (MBL: PM057), rabbit anti-ZEB1 (Cell Signaling: 3396), rabbit anti-SNAI1 (Cell Signaling: 3879), rabbit anti-SNAI2 (Cell Signaling: 9585), and mouse anti-actin (SigmaAldrich: A1978). Then, they were developed using secondary antibodies goat anti-rabbit HRP-conjugated (JacksonImmuno Research: 111-035-144) and goat anti-mouse HRP conjugated (SigmaAldrich: AP200P), respectively.