Rabbit anti sp1

The patient cohort used herein consists of 47 EC, 20 SEH, 12 CEH and 38 specimens of normal endometrium that underwent surgery at the First Affiliated Hospital of Anhui Medical University (AMU) (Hefei, Anhui, People's Republic of China) between 2001 and 2002 as previously described [58 (link)]. The Institutional Review Board of AMU approved the protocol for the use of patient specimens in this study [58 (link)]. Patient consent forms were obtained from all patients in accordance with the Declaration of Helsinki [58 (link)]. IHC analysis was performed as previously described [58 (link)] using rabbit anti-TFF3 was obtained from Abcam, Cambridge, MA; rabbit anti-SP1, rabbit anti-p-c-JUN and rabbit anti-c-JUN antibodies wereobtained from Cell Signaling Technology, Singapore [17 (link)]. The details of the cohort and IHC scoring methodology have previously been described in detail [30 (link), 58 (link), 59 (link)].

Pancreatic cancer cell lines (BxPC-3, Capan-1, SW1990, AsPC-1, HPAF-II, and HS-766T) and the normal human pancreatic ductal epithelial cell line, HPDE, were all obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). HS-766T cells were grown in 5% CO₂ with saturated humidity at 37 °C, cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mmol/L glutamine and 10% fetal bovine serum (FBS) (both from Gibco, Gaithersburg, MD, USA) and subcultured by harvesting with trypsin-EDTA. HPDE, BxPC-3, CAPAN-1, AsPC-1, and HPAF-II cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS. SW1990 cells were cultured in L-15 medium (Gibco) supplemented with 10% FBS. The 5-aza-2′-deoxycytidine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trichostatin A and 3-deazaneplanocin A (DZNeP) were purchased from Selleck Chemicals (Houston, TX, USA). Rabbit anti-Sp1, anti-EZH2, anti-Ago2, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-HDAC3 and anti-DNMT1 antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti-β-actin was purchased from Sigma-Aldrich.

Immunoblotting analyses were performed using 25 μg total protein from the cell lysates. Protein concentrations was determined using the Bradford protein assay (Bio-Rad). Electrophoresis was performed using either an 8% or 12% SDS-PAGE gel and then subsequently transferred to PVDF membranes (Bio-Rad). The blots were and reacted with mouse anti-S100B (1:2000, BD Biosciences, Catalog No. 612376), rabbit anti-Phopho-STAT3 (1:1000, Cell signaling, Catalog No. 9145), rabbit anti-STAT3 (1:1000, Cell signaling, Catalog No. 12640), mouse anti-CREB (1:1000, Cell signaling, Catalog No. 9104), rabbit anti-Phospho-CREB (1:1000, Cell signaling, Catalog No. 9198), GAPDH (1:10,000, Cell signaling, Catalog No. 5174), β-actin (1:10,000, Sigma, Catalog No. A5541), rabbit anti-SP1 (1:1000, Cell signaling, Catalog No. 9389), rabbit anti-Phospho-MEK1/2 (1:1000, Cell signaling, Catalog No. 9121), mouse anti-vinculin (1:5000, Sigma, Catalog No. V9131); rabbit phospho-AKT (S473) (1:1000, Cell signaling Catalog No. 9271). Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher) was used at dilutions recommended by the manufacturer to detect proteins levels.