Anti cd24 pe antibodies

Anti-PAK1, anti-pStat3 (Y705), and anti-JAK2 antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-p65, anti-Stat3, anti-β-actin, and anti-Lamin b were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-CD44-FITC and anti-CD24-PE antibodies were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Antibodies for ChIP and EMSA experiments were Anti-PAK1 and anti-pStat3. Human PAK1 siRNA and scrambled siRNA were purchased from Bioneer (Daejeon, Korea). The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Dissociated cancer cells were filtered through a 4 µm strainer and suspended in PBS supplemented with 2% FBS and 2 mM EDTA (FACS buffer) as previously described [73 (link)]. One µL of mouse IgG (1 mg/mL) was added and incubated at 4 °C for 10 min. The cells were then re-suspended in 1× binding buffer and anti-CD44 (APC) in combination with anti-CD24 (PE) antibodies (BD, Mississauga, ON, Canada) according to the manufacturer’s instructions for 30 min. The cells were washed twice with FACS buffer and 7-aminoactinomycin D (7-AAD, eBioscience, San Diego, CA, USA) and Annexin-V/V450 (BD) was added and incubated for 15 min at room temperature to assess dead and apoptotic cells. Flow cytometry was performed on the BD LSRFortessa. Data were analyzed with FlowJo software (Ashland, OR, USA).

Anti-GLI1, anti-GLI2, anti-SMO, and anti-SOX2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibody was obtained from Santa Cruz Biotechnology. Anti-CD44 FITC and anti-CD24 PE antibodies were obtained from BD Pharmingen (BD, San Jose, CA, USA). Human SMO- and SOX2-specific siRNAs were obtained from Bioneer Corp. (Daejeon, Korea).