Py416 c src

Experimental procedures of immunoblotting, co-immunoprecipitation, and purification of recombinant glutathione-S-transferase (GST)-tagged or non-tagged proteins were performed as described previously [19 (link), 34 (link)]. Immunoblotting was performed using antibody specific to human TRIP6, A20 (Bethyl Laboratories), pT183/Y185-JNK, pT202/Y204-ERK, pS32/36-IκBα, pS176/180-IKKα/β, pY416-c-Src, CYLD, IκBα, IKKβ, Histone H3 (Cell Signaling, Danvers, MA, USA), pT180/Y182-p38 (Promega, Madison, WI, USA), ERK, JNK, mouse TRIP6 (BD Biosciences, San Jose, CA, USA), HA (Cayman, Ann Arbor, MI, USA), FLAG (Sigma-Aldrich), GFP, TRAF6, p38, c-Src, GAPDH, GST, ubiquitin, or phosphotyrosine (Santa Cruz Biotechnology, Dallas, TX, USA).

Calu-3 in 3D culture were stained essentially as described for MDCK cysts [9 (link)]. Primary antibodies utilized were: Ki-67 (Epitomics, 2642-1), Cleaved Caspase 3 (Cell Signaling Technology, 9661L), JAM-A (BD Biosciences, 612120), Muc1 (Epitomics, 2900-1), GM130 (BD Biosciences, C65120), β-catenin (Santa Cruz, sc-7199), NHERF1 (Abcam, ab3452), Ezrin (BD Biosciences, 610603), Cleaved Caspase 8 (Cell Signaling Technology, 9496), β1-integrin (BD Biosciences, 610467), pY416-c-Src (Cell Signaling Technology, 6943P), pY1105-p190 (Abcam, ab55339). Alexa fluorophore-conjugated secondary antibodies (1:250) or Phalloidin (1:200) (both Invitrogen) and Hoechst to label nuclei (10 μg/ml), were utilized. Imaging was performed on a Zeiss 510 Confocal Microscope, using a 63x oil immersion lens. Image processing was performed using ImageJ. Images shown are representative of 3 separate experiments.