Anti cd44 15675 1 ap

Anti-RAD18 (MA532284) was from Invitrogen. StemLight™ Pluripotency Transcription Factor Antibody Kit (9093T) and Hippo Signaling antibody sampler kit (8579T) was from Cell Signaling Technology. Anti-CD44 (15675-1-AP) was from Proteintech. PMA (Sigma-Aldrich, MO, US) was dissolved in DMSO and stored at −20 °C. Anti-CD68 (25747-1-AP), anti-CD86 (13395-1-AP) and anti-CD163 (16646-1-AP) for immunofluorescence (IF) staining were purchased from Proteintech. APC Anti-CD86 (65165) and PE anti-CD163 (12-1639-41) were bought separately from Proteintech and eBioscience. The secondary antibody were bought from CWBIO (Beijing, China). Anti-human TGF-β1 antibody (21898-1-AP) and neutralizing antibodies (69012-1-Ig) were purchased from Proteintech.

Harvested cells were lysed in RIPA buffer, and protein concentrations were quantified using BCA protein assay kits (Beyotime, China). Total protein lysates from each sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, USA). The membranes were blocked in Tris-buffered saline with 5% non-fat milk and then incubated 4 °C overnight with anti-MUC16 (sc-365002, Santa Cruz Biotechnology, USA), anti-CD133 (sc-365537, Santa Cruz), anti-CD44 (15675-1-AP, Proteintech, USA), and anti-Tubulin (MA5-16308, Thermo Fisher Scientific, USA) antibodies diluted in 5% bovine serum albumin (BSA). All horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Sigma (USA) and used at a working dilution of 1:5000 in working solution. Blots were visualized with chemiluminescent HRP substrate (Millipore, USA). All the antibodies are listed in Additional file 8: Table S3.