Anti cd133

Harvested cells were lysed by RIPA buffer. After sonication, samples were centrifuged at 12 000 g for 15 min at 4°C. Total protein concentration was determined by applying DC Protein Assay Kit I (Bio‐Rad, USA). Proteins were transferred to Hybond nitrocellulose membranes (USA) after separation on 12% SDS‐PAGE. 5% skim milk was added to seal the membrane in Tris‐buffered saline (pH 7.5) at room temperature. The membrane was incubated with the primary antibody overnight at 4°C, followed by 4‐h incubation with the secondary antibody at room temperature. Protein bands were developed by ECL kit (Millipore, USA). Protein levels were assessed by ImageJ (USA). Primary antibodies including anti‐CD133, anti‐CD44, anti‐Oct‐4, anti‐HK2, anti‐PKM2, anti‐LDHA, anti‐β‐actin, and secondary antibody anti‐IgG were purchased from Abcam (UK).¹⁶

The following antibodies were purchased: for Western blotting, anti-β-catenin (1:5000; Abcam, cat. no. ab32572), anti-β-actin (1:5000; Bioss, cat. no. bs-0061R), anti-CDK5RAP2 (1:2000; Abcam, cat. no. ab70213), anti-survivin (1:2000; Abclonol, cat. no. A1551), anti-ALDH1 (1:1000; Cell Signaling Technology, cat. no. 54135), anti-Notch1 (1:1000, Abcam, cat. no. ab52627), anti-EZH2 (1:1000; Cell Signaling Technology, cat. no. 5246), anti-CCND1 (1:1000, Cell Signaling Technology, cat. no. 55506), and horseradish peroxidase-conjugated secondary antibodies (1:1000; Cell Signaling Technology, Inc.); for IHC analyses, anti-CDK5RAP2 (1:400; Abcam, cat. no. ab235893), anti-ALDH1 (1:50; Abcam, cat. no. ab52492), anti-SOX2 (1:100; Abcam, cat. no. Ab92494), anti-CD44 (1:4000; Abcam, cat. no. ab189524), anti-CD133 (1:1000; Abcam, cat. no. ab222782), anti-Notch1 (1:150; Abcam, cat. no. ab52627), anti-EZH2 (1:200; Cell Signaling Technology, cat. no. 5246), and anti-CCND1 (1:200; ABclonal, cat. no. A19038); and for immunofluorescence analyses, anti-α-tubulin (1:500, YL1/2; Santa Cruz Biotechnology, cat. no. sc-53029), anti-γ-tubulin (1:1000, GTU88; Sigma-Aldrich, cat. no. T5326), and Alexa Fluor-conjugated secondary antibodies (1:500; Thermo Fisher Scientific).

Cells were lysed in lysis buffer. Equal amounts of total proteins were loaded onto 4-12% SDS–PAGE gels and transferred to PVDF membranes (GE Healthcare Life Sciences, NJ, USA). The membranes were blocked with 5% milk dissolved in TBS containing 0.02% Tween 20 and incubated overnight at 4°C with specific primary antibodies. The membranes were subsequently incubated with specific horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a Fusion FX5 system ((Vilber Lourmat, France). The following primary antibodies were used: anti-cleaved caspase 3, anti-E-cadherin, anti-cleaved caspase 9, anti-cleaved PARP1 (Cell Signaling Technology), anti-SNAIL1, anti-Vimentin, anti-Twist, anti-Slug, anti-Zeb1, anti-Nanog, anti-Sox2, anti-CD44 (Santa Cruz), anti-N-cadherin and anti-Oct4 (BD Biosciences), anti-Survivin, anti-CD133 (Abcam), anti-ALDH (Avivasysbio), and anti-β-actin (Sigma).

AQP3/CD133/CD44/CD90/EPCAM expression was analyzed in paraffin-embedded specimens obtained from 120 patients. Four-µm-thick tissue sections were deparaffinized in xylene and dehydrated before antigen retrieval for 5 min using an autoclave. The endogenous peroxidase activity was blocked using hydrogen peroxide (0.3%), and non-specific immunoglobulin binding sites were blocked by normal goat serum for 30 min at 37 °C. Tissue sections were incubated with anti-AQP3 (1:1000, Abcam), anti-CD133 (1:200, Abcam), anti-CD44 (1:1000, Abcam), anti-EPCAM (1:200, Abcam), and anti-CD90 (1:200, Abcam) overnight at 4 °C. Then, the sections were incubated with biotinylated goat anti-rabbit IgG as a secondary antibody (Maixin Kit, China) for 1 h at room temperature, followed by incubation with streptavidin–biotin horseradish peroxidase-conjugated (Maixin Kit) for 30 min at room temperature. The peroxidase reaction was developed with 3′-diaminobenzidine tetrahydrochloride (Maixin Kit). The expression levels of the proteins were scored semi-quantitatively according to the percentage of positively stained cells combined with the staining intensity according to our previous study^{18 (link)}. The specimens were assessed three times.