Ps536 nf κb p65

Antibodies against Stat3 (#9139; 1/1500 for western blot analysis, 1/800 for immunofluorescence), pY705-Stat3 (#9145; 1/1000 for western blot analysis, 1/100 for immunohistochemical staining), NF-κB p65 (#8242; 1/1000 for western blot analysis, 1/1600 for immunofluorescence), and pS536-NF-κB p65 (#3033; 1/1000) were purchased from Cell Signaling Technology; ERK1/2 (#AF0155; 1/1,000), and pT202/Y204-ERK1/2 (#AF1015; 1/1,000 for western blot analysis, 1/200 for immunofluorescence) were purchased from Affinity Biosciences. Antibodies against LIF (#ab2020; 1/200 for immunohistochemical staining) and CD44 (ab216647) were purchased from Abcam. CD90 (66766-1-Ig) and β-actin (#66009-1-Ig; 1/5000) were purchased from Proteintech.

Cells were grown to ~80–90% confluence prior to harvest and lysis. Western blot analysis on whole cell lysates was performed as described previously [36 (link)]. Nuclear extracts for detection of transcription factors were prepared as previously described [37 (link)]; 25 μg of protein were prepared for each samples. Primary antibodies to EGFR (#2232), MET (#3127), pY1234⁄Y1235-MET (#3129), Y1289-Her3 (#4791), pT705-STAT3 (#9131), pS536-NFκB-p65 (#3031), pS465/467-SMAD2 (#3101), Akt (#9272), pS473-Akt (#9271), Erk (#9102), pT202/T204-Erk (#4377), and PARP (#9542) were obtained from Cell Signaling (Beverly, MA). Primary antibodies to pY1068-EGFR (#44-788G) were obtained from Invitrogen. Primary antibodies to Vimentin (BDB550513) were purchased from BD Biosciences (San Diego, CA). Primary antibodies to ZEB1 (sc25388), fibronectin (sc29011), Her3 (sc285), E-cadherin (sc8426), N-cadherin (sc7939), PTEN (sc7974), Snail (sc28199), Slug (sc15391), Twist (sc6269), and Lamin A/C (sc20681) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies to β-actin (SigmaA-1978) were purchased from Sigma-Aldrich. Horseradish peroxidase-conjugated goat anti-mouse (NXA931) and anti-rabbit (NA934V) secondary antibodies were obtained from Amersham Biosciences (Piscataway, NJ).