Anti xiap

The following primary antibodies were used for Western blotting: polyclonal anti-cIAP-1 (R&D Systems, AF8181), polyclonal anti-cIAP-2 (R&D Systems, AF8171), polyclonal anti-XIAP (R&D Systems, AF8221), and polyclonal anti-FADD (Cell Signaling Technology, 2782). Whole-cell lysates were collected and lysed in NP-40 buffer (Invitrogen, FNN0021) supplemented with 1% Halt protease inhibitor and 1% Halt phosphate inhibitor cocktails from ThermoFisher Scientific (Waltham, MA). Lysates were vortexed and frozen at − 80 °C prior to use. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Equal amounts of lysates (15 µg) for each cell line were loaded into 4–12% gradient Bis–Tris gels (Invitrogen) and transferred to PVDF membranes for 6 min using the iBlot system (Invitrogen), according to the manufacturer’s standard protocol. Membranes were blocked for one hour in Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE) and incubated with the primary antibody overnight at 4 °C. Membranes were washed before incubation with species-appropriate Odyssey secondary antibodies for 1 h at room temperature. Quantification of the protein expression detected by Western blotting was done by protein densitometry normalized to actin with ImageJ (National Institutes of Health, Bethesda, MD).

Following antibodies were used: anti-p21 (1:500), anti-vimentin (1:5,000), anti-E-cadherin (1:250), anti-survivin (1:2,000), anti-phospho-Erk1/2 (1:1,000), and anti-Erk1/2 (1:1,000)were from Cell Signaling Technology (Boston, MA); anti-N-cadherin (1:4,000) was from EMD Millipore (Billerica, MA); anti-β-catenin (1:250) and anti-XIAP (1:1,000) were from R&D Systems (Minneapolis, MN); and anti-β-actin (1:3,000) was from Santa Cruz Biotechnology (Dallas, TX).