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4 protocols using anti survivin 71g4b7

1

Protein Solubilization and Immunoblotting

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Protein solubilization and immunoblotting were conducted as previously described [15 (link)]. The following commercially available antibodies were used: anti-TAZ (V386) (#4883; Cell Signaling Technology, Beverly, MA, USA), anti-survivin (71G4B7) (#2808; Cell Signaling Technology), anti-p53 (sc-126; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p21 (F-5, Santa Cruz Biotechnology), and anti-β-actin-HRP (C4; Santa Cruz Biotechnology).
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2

Apoptosis Regulators Expression Analysis

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Anti-BIRC6 (Novus Biologicals, #NB110-40730) [28 (link)], anti-survivin (71G4B7) (Cell Signaling, #2808) [49 (link)]; anti-XIAP (H-202) (#sc-11426, Santa Cruz Biotechnology, Santa Cruz, CA) [50 (link)]. cIAP-1/HIAP-2 antibody (R&D Systems #MAB8181) for IHC, anti-cIAP1 (D5G9) (#7065, Cell Signaling Technology, Danvers, MA) for Western Blotting. The same antibodies were used for immunohistochemistry and Western blotting unless otherwise indicated.
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3

Antibodies for Western Blot and Immunofluorescence

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The antibodies used in this study were obtained from the following sources: anti-survivin (71G4B7, Cat#2808), anti-XIAP (3B6, Cat#2045), anti-phospho-JAK1 (Y1034/1035; Cat#3331), anti-phospho-STAT3 (Y705; D3A7, Cat#9145), anti-cleaved PARP (Cat#5625T), anti-cleaved caspase 3 (Cat#9664) and anti-phospho-JAK2 (Y1007/1008; Cat#3771) from Cell Signaling Technology Inc. (Beverly, MA, USA); anti-actin (I-19, Cat#sc-1616), anti-GAPDH (0411, Cat#sc-47724), anti-JAK1 (B-3, Cat#376996), anti-phospho-STAT3 (Y705; B-7, Cat#sc-8059), anti-STAT3 (F-2, Cat#sc-8019), from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); anti-vinculin (Cat#V-4505) from Sigma-Aldrich. Antibody binding was detected with donkey anti-mouse IgG-HRP (Cat#A16017), donkey anti-rabbit IgG-HRP (Cat#A16029) and donkey anti-goat IgG-HRP (Cat#A15999) from Thermo Fisher Scientific Inc. (Waltham, MA, USA); Alexa Fluor™ 488-conjugated donkey anti-mouse (Cat#A31572, Molecular Probes, Eugene, OR, USA) was used for antibody binding detection in immunofluorescence assays.
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4

Western Blotting for Protein Expression

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Western blotting for the expression of E-cadherin, vimentin, CD44, LC3A, MET, ATG5, total EGFR, phosphorylated EGFR, poly(ADP-ribose) polymerase-1 (PARP-1), γH2AX, total AKT, phospho-AKT, total ERK1/2, phospho-ERK1/2, Mcl-1, Bim, and β-actin was performed as described previously. 6, 13 Additional primary antibodies for immunoblotting were anti-ZEB1 (HPA027524; 1:1000 dilution; Atlas Antibodies, Stockholm, Sweden), anti-ATG7 (D12B11; 1:1000 dilution; Cell Signaling), anti-Pin1 (1:1000 dilution; Cell Signaling), anti-Survivin (71G4B7; 1:1000 dilution; Cell Signaling), and anti-Puma (EP512Y; 1:2000 dilution; Abcam Japan, Tokyo, Japan). Band intensity levels on X-ray films were normalized to β-actin using the Image J software (National Institutes of Health, Bethesda, MD, USA).
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