Peroxiredoxin 1

Normal Human Pancreatic Ductal Epithelial (HPDE) cells and mutant Kras HPDE cells (HPDE-Kras) were obtained from the Tsao lab [45 (link), 46 (link)]. Panc1 (primary tumor cells) and AsPC-1 (metastatic cells) were obtained from ATCC. HPDE and HPDE-Kras cell lines were cultured and treated in low serum keratinocyte serum free media (supplied supplements- 1ml bovine pituitary extract, 1ul EGF) (Gibco, Waltham, MA, Cat#10724-011). Panc1 and ASPC-1 cells were cultured in full DMEM media (10% FBS) and treated in serum free DMEM. All in vitro and primary culture treatments were 24 hrs. Auranofin was from Sigma (St. Louis, MO) (1uM Auranofin).
Antibodies- Western and IP: Peroxiredoxin-1 (Abcam, Cambridge, MA, Cat# 59538), Phospho-Prdx1 (Tyr194) (Cell Signaling (CST), Danvers, MA, Cat#14041), Thioredoxin-1 (CST Cat#2429), Sulfiredoxin (Proteintech group (PTG), Rosemont, IL, Cat#14273-1-AP), anti-dimedone (CSA) (Millipore, Billerica, MA, Cat# 07-2139); pERK, ERK, pAKT, AKT (CST), GAPDH (Santa Cruz Biotechnology, Dallas, TX), Conformation specific secondary anti-rabbit IgG (HRP conjugated) (CST Cat#5127). IF and IHC: Peroxiredoxin-1 (Abcam Cat#41906), Anti-Thioredoxin / TRX antibody (Abcam Cat#86255), Sulfiredoxin (PTG Cat# 14273-1-AP), CK19 (Cytokeratin 19) -TROMA III Ab (DSHB, University of Iowa, IA).

Hippocampal MF and CF proteins from 8-week-old male C57BL/6 mice treated with ketamine for 2 h, 14 h, 24 h and 72 h were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot was performed based on standard protocols. After electrophoresis, proteins were transferred to PVDF membranes (Immobilon-P, Millipore, Billerica, USA). Primary antibodies were against adenosinmonophosphate-activated protein kinase (AMPK, Abcam, Cambridge, UK), phosphorylated AMPK (pAMPK, Cell Signaling, Merck, Darmstadt, Germany), peroxiredoxin 1 (Prdx1, Abcam, Cambridge, UK), peroxiredoxin 3 (Prdx3, Abcam, Cambridge, UK). Anti-rabbit, anti-mouse and anti-goat ECL horseradish peroxidase-linked secondary antibodies (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) were used. The densitometric analyses were performed with the Image Lab software (Bio-Rad, Munich, Germany).