Horseradish peroxidase hrp conjugated anti mouse and anti rabbit secondary antibodies

Alpha modification of Eagle's medium (AMEM), phosphate‐buffered saline (PBS), trypsin, and antibiotic–antimycotic preparations were purchased from Wisent (St Bruno, Quebec, Canada). Fetal bovine serum (FBS), horse serum (HS), Lipofectamine RNAiMAX, and Opti‐MEM 1X Reduced Serum Medium were purchased from Thermo Fisher Canada (Burlington, Ontario Canada). Amino acid‐free RPMI 1640 medium was purchased from US Biologicals (Salem MA). Sodium 4‐methyl‐2‐oxovalerate (sodium salt of KIC), 2‐deoxyglucose, protease and phosphate inhibitor cocktails, anti‐BCAT2 and anti‐gamma tubulin antibodies, siRNA oligonucleotides, amino acid standard, o‐Phthalaldehyde, interleukin‐6, and homocysteine were purchased from Sigma Aldrich (Oakville, Ontario, Canada). Phospho (ph) ribosomal protein S6 kinase 1 (S6K1) (T389), ph‐ribosomal protein S6 (S6) (S235/236), ph‐Akt (S473), ph‐SAPK/JNK (T183/Y185), ph‐glycogen synthase (S641), BCKDH‐E1α, horseradish peroxidase (HRP)‐conjugated anti‐rabbit and anti‐mouse secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). [³H]‐2‐deoxyglucose was purchased from Perkin Elmer (Markham, Ontario, Canada) while chemiluminescence substrate was from Millipore (Etobicoke, Ontario, Canada). TNF‐α was purchased from Shenandoah Biotechnology (Warwick, PA).

PTNs were a generous gift from Won-Kyo Jung, Pukyong National University, Busan, South Korea. PTNs were prepared from Ecklonia cava collected along the Jeju Island coast of Korea as previously described [43 (link)]. Composition and chemical structures of PTNs in the ethanolic extracts were previously characterized [44 (link),45 (link)]. Easyef^TM, a commercial spray-type ointment containing rhEGF, was purchased from Daewong Pharmaceuticals (Seoul, South Korea). The primary antibodies used were as follows: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#5174), anti-p65-NF-κB (#8242), anti-IL-1β (#12242), and anti-HO-1(heme oxygenase) (#5853) purchased from Cell Signaling Technology (Danvers, MA, USA); anti-ASC (SC514414) and anti-NRF2 (SC1722) purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-AQP3 (AB3276) purchased from Merck Millipore (Burlington, MA, USA); and anti-COX2 (#610203) purchased from BD Biosciences (Franklin Lakes, NJ, USA). Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit and anti-mouse antibodies were purchased from Cell Signaling Technology.

SH-SY5Y (ECACC, Salisbury, Wiltshire, United Kingdom), HCT-15, HCT116, HEK293, HEK293T and MCF-7 cells were cultured in DMEM/F-12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum. To obtain normal human matured neuronal cells in a culture system, ReproNeuro, a neuron progenitor derived from human iPS cells, was purchased from ReproCELL (Yokohama, Japan) and maintained in ReproNeuro maturation medium for 14 days according to the manufacturer’s instructions. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 6-hydroxydopamine hydrobromide (6-OHDA), cadmium chloride (CdCl₂), rotenone, tert-Butylhydroquinone (tBHQ), sulforaphane and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paraquat, caffeine and curcumin were purchased from Wako Chemicals (Osaka, Japan). The antibodies used were as follows: an antibody against PINK1 (rabbit monoclonal, 6946), an antibody against NRF2 (rabbit monoclonal, 12721), horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA, USA), and an antibody against Tubulin (mouse monoclonal, T5168; Sigma-Aldrich).

17β-estradiol (E2) was purchased from Sigma Aldrich (Saint Louis, MO, USA). TCDD and NNK were obtained from Toronto Research Chemicals Inc. (TRC, North York, Ontario, Canada). E2 was dissolved in corn oil, and TCDD was dissolved in dimethylsulfoxide (DMSO) and stored in the dark at −20°C until use. NNK was dissolved in normal saline solution immediately before use. Primary antibodies against IκB, phospho-IκB, NFκB subunit p65, p50, COX-2, cyclin D1, ERK1/2, phospho-ERK1/2, JNK, phospho-JNK, p38, phospho-p38, PCNA, iNOS, MMP-9, GAPDH, and horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Cardiac tissues were homogenized with RIPA lysis buffer to prepare lysates. Protein samples were subjected to SDS-PAGE, and transferred onto a PVDF membrane (Bio-Rad, Hercules, CA). After blockade with 5% skimmed milk, membranes were incubated with antibodies. Primary antibodies to fibronectin, CTGF, TNF-α, 3-NT, 4-HNE, Nrf2, and HO-1 were obtained from Abcam (Cambridge, MA); collagen1A1, ANP, IL-1β, NQO1, CAT, SOD2, and β-actin were obtained from Santa Cruz Biotechnology (Santa, CA); PAI-1 was obtained from BD Biosciences (Franklin Lakes, NJ); phosphorylated NF-κB and total NF-κB, as well as horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA). Western blot images were acquired using ChemiDoc Touch Imaging System (Bio-Rad). Grayscale values of bands were analyzed using the Image Lab software (Bio-Rad); protein expressions were normalized relative to those of β-actin.