Ac 15

Cells were grown in 10-cm dishes, exposed to treatments where required and then lysed as previously described [54 (link)]. Equal amounts of whole-protein extract were resolved on a 8–10% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Biosciences, Merck Life Science), which was probed with primary antibodies against RAGE (MAB1145, R&D Systems, 1:1000), EphA3 (12,480–1-AP, Proteintech, 1:500), SP1 (1C6, Santa Cruz Biotechnology, 1:1000) and β-actin (AC-15, Santa Cruz Biotechnology, 1:4000) and then revealed using the Clarity™ Western ECL Substrate (Bio-Rad).

Total cell lysates were performed by lysing cells in a 10× RIPA buffer containing 20 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1 mM Na₂ EDTA 1 mM EGTA 1% NP-40 1% sodium deoxycholate 2.5 mM sodium pyrophosphate 1 mM β-glycerophosphate 1 mM Na₃ VO₄ 1 µg/mL leupeptin and protease inhibitor mixture. Protein concentrations were then measured using Bio-rad protein assay protocol. Lysates are resolved on a 7.5% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. The following antibodies are used: from Santa Cruz (Dallas, TX, USA): RICK (H-300); RICK (A-10); PCNA Antibody (PC10); Actin (AC-15); ERK 1 Antibody (K-23); and ERK 2 Antibody (C-14). From cell signaling technology (Danvers, MA, USA): phospho-RIP2 (Ser176) (E1I9J) #14397; NF-κB p65 (D14E12) #8242; Phospho-NF-κB p65 (Ser536) (93H1) #4887. RIPK2 Phospho-Y474 polyclonal antibody was obtained from a MediMab production order from the Baksh lab (University of Alberta, Edmonton, AB, Canada). For immunoblots, densitometry was performed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) of the scanned image using region of interest analysis. All results were normalized to normal breast tissue.