Secondary abs

Carcinoma cells were seeded at a density of 8 × 10⁵ (SiHa), 5 × 10⁵ (HeLa), or 1 × 10⁶ (SW756) cells/6‐well culture dish. 24 h later, they were incubated with medium or conditioned media of Th17 cells (diluted 1 : 4) for 15 min. To prepare whole cell extracts, cells were lysed with 2× lysis buffer (130 mm Tris/HCl, 6% SDS, 10% 3‐Mercapto‐1,2‐propanediol, 10% glycerol) and 3 times treated with ultrasound for 5 s. 15 µg of the whole protein extracts was separated by SDS gel electrophoresis and transferred to a nitrocellulose membrane (Whatman, GE Healthcare, Freiburg, Germany). Rabbit anti‐pThr308‐AKT (1 : 1000; D25E6; Cell Signaling), rabbit anti‐pSer473‐AKT (1 : 2000; D9E; Cell Signaling), rabbit anti‐AKT1 (1 : 1000; C73H10; Cell Signaling), rabbit anti‐AKT2 (1 : 1000; D6G4; Cell Signaling), rabbit anti‐AKT3 (1 : 1000; E1Z3W; Cell Signaling), rabbit anti‐panAKT (1 : 1000; C67E7; Cell Signaling), and mouse anti‐β‐Actin antibody (1 : 5000; AC‐15; Sigma‐Aldrich) were used. Secondary Abs (Sigma‐Aldrich) and SuperSignal West Dura Substrate (Thermo Fisher Scientific, Schwerte, Germany) were used for detection with ChemiDoc XRS+ Molecular Imager. Expression was quantified with the Image Lab software (both Bio‐Rad, Feldkirchen, Germany).

Abs to HOXA9 were purchased from Millipore (for Western blot) and Santa Cruz Biotechnology (for chromatin IP). Abs to P-cadherin were purchased from BD Biosciences (for Western blot) and Abcam (for neutralization). Other Abs were as follows: E-cadherin (Invitrogen), N-cadherin, active caspase-3 (BD Biosciences), actin, secondary Abs (Sigma-Aldrich). HOXA9 cDNA was provided by Corey Largman (Veterans Affairs Medical Center, San Francisco). CDH3 cDNA and pGFP-V-RS plasmids containing HOXA9, CDH3 and non-targeting shRNAs were purchased from OriGene Technologies.