Anti sox2 antibody

Protein extraction and western blotting analysis were performed using previously standard procedures (17 (link)). The following antibodies were used for western blotting: anti-E-cadherin antibody(#20874-1-AP Proteintech, China), anti-N-cadherin antibody(#22018-1-AP Proteintech, China), anti-ZEB1 antibody(#66279-1-Ig Proteintech, China), anti-Vimentin antibody(10366-1-AP Proteintech, China), anti-SOX2 antibody(#11064-1-AP Proteintech, China), anti-GAPDH antibody(#10494-1-AP Proteintech, China), and anti-PD-L1 antibody(#66248-1-Ig Proteintech, China).

NPCs markers SOX2 and Nestin were detected by imaged-based immunocytochemistry analyses. Cells were plated onto Matrigel-coated (Corning) 12-well plates on cover slips at a density of 0.4 × 10⁶ cells per well. After 2 days, cells were fixed using 4% paraformaldehyde, permeabilized with 0.1% Triton-X in phosphate-buffered saline (PBS), and blocked with 2.5% normal donkey serum (Abcam) in PBS/Tween 20. SOX2 protein was detected using anti-SOX2 antibody (ProteinTech Group) diluted to 1:500, and Nestin protein was detected using anti-Nestin antibody (Sigma-Aldrich) diluted to 1:200, following 1-h incubation at room temperature. Cells were incubated with Alexa Fluor 488 antibody (Thermo Fisher Scientific) diluted to 1:2,000 for Nestin detection and with Alexa Fluor 568 antibody (Thermo Fisher Scientific) diluted to 1:1,000 for SOX2 detection at room temperature for 45 min. Prepared coverslips were mounted onto slides using ProLong™ Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) and imaged using a Nikon A1R confocal microscope (UAB High Resolution Imaging Facility).

Serial sections of a human lung tumor tissue array containing 116 NSCLC tumor tissue samples (60 squamous cell carcinoma and 56 adenocarcinoma specimens) and 16 normal/normal adjacent lung tissue samples (N/NAT) (4N and 12 NAT specimens) were purchased from Alenabio (LC121c and LC241h). H&E and immunohistochemical staining were performed according to previously described protocols,^{42 (link)} using a 1:1000 dilution of an anti-WIP1 antibody (Abcam, ab31270), 1:100 dilution of an anti-phospho-p38 MAPK antibody (Cell Signaling Technology, #4511), 1:100 dilution of an anti-SOX2 antibody (Proteintech, 11064-1), and 1:100 dilution of an anti-ALDH1A1 antibody (Santa Cruz, sc-374149). The images were acquired with an optical microscope fitted with a camera (Olympus, Japan). To evaluate the expression of WIP1, p-p38, SOX2, and ALDH1 in normal or tumor tissue samples, staining scores were evaluated by multiplying the positive staining area (scored as 1: 0–25%; 2: 26–50%; 3: 51–75%; 4: 76–100%) with the staining intensity (scored as 1: weak; 2: moderate; 3: strong; 4: very strong).