Nuclear extract kit

Garlic extract was obtained from Egarak (Busan, Korea). Polyclonal antibodies against p-Cdc2 p34 (sc-12340-R), Cdc2 p34 (sc-54), CHK2 (sc-9064), Cdc25c (sc-327), p-Cdc25c (sc12354), p21WAF1 (sc-756), p53 (sc-126), Cyclin A (sc-751), Cyclin B1 (sc-245), p-ATM (sc-47739), ATM (sc-23921), WEE1 (sc-325), HSPA6 (sc-374589) and GAPDH (sc-20357) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Polyclonal antibodies against CHK1 (2360), p-CHK1 (2341), p-CHK2 (2661), ERK (9102), p-ERK (9101), JNK (9258), p-JNK (9251), p38 MAP kinase (9212), p-p38 MAP kinase (9211), AKT (9272), and p-AKT (9271) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Goat anti-rabbit IgG-horseradish peroxidase (HRP) (sc-2004), goat anti-mouse IgG-HRP (sc-2005), and donkey anti-goat IgG-HRP (sc-2020) were purchased from Santa Cruz Biotechnology Inc. Western Lightning Plus-ECL was obtained from PerkinElmer, Inc. (PerkinElmer, MA, USA). U0126, SB203580, SP600125, and wortmannin, were obtained from Calbiochem (San Diego, CA). A Nuclear Extract kit and EMSA Gel Shift kit were obtained from Panomics (Fremont, CA, USA). HSPA6 cDNA was obtained from the Korea human gene bank.

Hydrangenol was purchased from Coresciences Co. (Seoul, South Korea). Antibodies against CDK2, CDK4, cyclin D1, cyclin E, p21^WAF1, p27^KIP1, p53, and GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Total ERK, p38, JNK, AKT, phospho-ERK, phospho-p38, phospho-JNK, and phospho-AKT antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The polyclonal MMP-9 antibody was purchased from Chemicon (Temecula, CA, USA). The nuclear extract kit and the electrophoretic mobility shift assay (EMSA) gel shift kit were obtained from Panomics (Fremont, CA, USA). The p38-specific inhibitor SB203585 was purchased from Calbiochem (San Diego, CA, USA).

Nuclear extracts were prepared using Nuclear Extract Kit (Panomics, CA, USA) according to the manufacturer’s instructions. The concentration of nuclear protein was determined using the bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL) to normalize for the amounts of protein within each experiment. TF array analysis (Panomics) was used to profile activities of 345 TFs. Any spots with a two-fold increase or decrease are considered significant.

Resveratrol (≥98%, analytical standard grade) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Polyclonal antibodies against extracellular signal-regulated kinase ERK (9102S), phospho-ERK (9101S), p38 mitogen-activated protein kinase (9212S), phospho-p38 MAPK (9211S), Jun N-terminal-kinase (9258S), phospho-JNK (9251S), AKT (9272S), phospho-AKT (9272S), and p21WAF1 (2947S) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Polyclonal antibodies against CDK2 (sc-163), cyclin E (sc-377100), CDK4 (sc-23896), cyclin D1 (sc-8396), p53 (sc-1641), p27KIP1 (sc-1641), β-actin (sc-47778), 5α-reductase 2 (sc-293232), AR (sc-7305), FGF-2 (sc-1360), B-cell lymphoma-2 (sc-7382) and Bcl-2-associated x protein (sc-20067) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). A nuclear extract kit and electrophoretic mobility shift assay (EMSA) gel shift kit were obtained from Panomics (Fremont, CA, USA). Polyclonal antibodies against PARP-1 (sc-7150), caspase-9 (sc-7885), caspase-7 (9492S), caspase-3 (sc-7148), cleaved PARP-1 (sc-7150), cleaved caspase-9 (sc-7885), cleaved caspase-7 (9492S), cleaved caspase-3 (sc-7148), and XIAP (sc-11426) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The DNA binding activity of STAT5b was assessed using EMSA, in which a labeled double-stranded DNA was used as a DNA probe to bind active STAT5b proteins in nuclear extracts. Nuclear protein extracts were prepared with a nuclear extract kit (Panomics, AY2002). The EMSA experiment was performed by incubating a biotin-labeled transcription factor-STAT5b probe with treated and untreated nuclear extracts. Proteins were resolved on a non-denaturing 6 % PAGE gel (Bio-Rad, Korea). The proteins in the gel, transferred to a nylon membrane and detected using streptavidin-HRP and a chemiluminescent substrate.

DATS (SMB00289) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Cell Signaling Technology (Danvers, MA, USA). The nuclear extract kit and electrophoretic mobility shift assay (EMSA) gel shift kit were obtained from Panomics (Fremont, CA, USA). cDNAs of ANGPTL4, PLCXD1, MMP3, and vectors pOTB7 and pCNS were obtained from the Korean Human Gene Bank. The human bladder carcinoma cell line EJ was purchased from the American Type Culture Collection (Manassas, VA, USA). Detailed information on cells and materials is available in the Supporting Information.​​

Anti-extracellular signal-regulated kinase (ERK), anti-phospho-ERK, anti-p38 mitogen-activated protein kinase (MAPK), anti-phospho-p38 MAPK, anti-Janus kinase (JNK), anti-phospho-JNK, anti-protein kinase B (AKT), and anti-phospho-AKT antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Polyclonal anti-cyclin D1, anti-cyclin E, anti-cyclin-dependent kinase (CDK) 2, anti-CDK4, anti-p53, anti-p21WAF1, anti-p27KIP1, and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Moreover, a Ki-67-specific antibody was obtained from Invitrogen (Waltham, MA, USA). Moreover, a nuclear extract kit and electrophoretic mobility shift assay (EMSA) gel shift kit were purchased from Panomics (Fremont, CA, USA). SP600125 were purchased from Calbiochem (San Diego, CA, USA).