Ab284389

Our study homogenized cells in ice-cold lysis buffer and centrifuged at 12,000×g for 20 min and at 4 °C. Lysates (50 μg protein/lane) went through separation by 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA. USA). Subsequently, we blocked the membranes utilizing 5 % BSA for 1 h, cut them per each protein molecular weight, and incubated them incubation at 4 °C with the corresponding primary antibodies for a whole night. Our study employed these primary antibodies: anti-LGR5 antibody (ab75850, Abcam), anti-CD133 (ab284389, Abcam), anti-ac⁴C (ab252215, Abcam), anti-beta Actin (ab8226, Abcam), and anti-NAT10 (ab194297, Abcam) antibodies. Subsequently, the membranes were investigated utilizing horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (A0208, Beyotime, Jiangsu, China) at room temperature for 2 h employing an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA), we visualized protein bands per the protocols. ACTB was utilized as a loading control, performing protein expression quantification using densitometry.

Slices of tissues were subjected to antigen retrieval by heating in a microwave oven in 10 mmol/L citrate buffer for 3 min. Then, sections were incubated with primary antibodies against Ki-67 (ab16667, Abcam), FOXQ1 (MA5-17078, ThermoFisher) and CD133 (ab284389, Abcam) at 4°C overnight. Afterward, slices were incubated with poly-peroxidase-anti-mouse/rabbit IgG and detected using DAB (Bioss, Beijing, China). Hematoxylin-based reagents were used to create the immunohistochemistry response, which was subsequently seen under a microscope.