Donkey anti rabbit igg hrp

EF24, N-acetylcysteine (NAC), catalase and rhTrxR1 protein were purchased from Sigma (St. Louis, MO). Erastin was purchased from Selleck Chemicals. Human gastric cancer cell lines SGC-7901, BGC-823 and KATO III, normal Human Gastric Epithelial Cell Line (GES-1) and normal Rat Kidney Proximal Cell Line (NRK-52E) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were routinely cultured in RPMI 1640 medium (Gibco, Eggenstein, Germany) containing 10% heat-inactivated fetal bovine serum (Gibco, Eggenstein, Germany), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified cell incubator with an atmosphere of 5% CO₂ at 37°C. Antibodies including anti-Bcl-2, anti-Bax, anti-cleaved PARP, anti-TrxR1, anti-GAPDH, goat anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies including anti-CHOP, anti-ATF4, anti-p-eIF2α and anti-eIF2α were purchased from Cell Signaling Technology (Danvers, MA). FITC Annexin V apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ).

The microdissected growth plate obtained as described above was homogenized in 50 µl of Laemmli sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% (v/v) glycerol, 0.01% bromophenol blue) and the protein content was determined with the BCA Protein Assay (Pierce). Samples were then reduced in 5% β-mercaptoethanol, separated by 7% SDS–PAGE and blotted on a polyvinylidenedifluoride membrane (PVDF, Hybond). Primary antibodies used in this study were: anti-β-catenin, anti-cyclin D1, and anti-p107 (sc-7199, sc-753, and sc-318 respectively, Santa Cruz Biotechnology, Inc.), anti-p130 (610261, BD Transduction Laboratories™), anti-phospho-β-catenin (Ser33/37/Thr41, 9561, Cell Signaling Technology), and anti-β-actin (A5316, Sigma–Aldrich). The following secondary HRP-conjugated antibodies were used: donkey anti-rabbit IgG-HRP (sc-2077, Santa Cruz Biotechnology, Inc.) and sheep anti-mouse IgG-HRP (NA931, GE Healthcare). Four percent milk powder in 1× TBS-T (20 mM Tris–HCl, pH 7.5, 0.5 mM NaCl, and 0.1% (v/v) Tween-20) was used for blocking and for diluting antibodies. Chemiluminescence detection was achieved with the ECL Plus Western blotting detection reagents (GE Healthcare) and autoradiography films (Hyperfilm, GE Healthcare). Films were digitalized by VersaDoc™ Imaging System (Bio-Rad) and densitometric analysis was performed by Quantity One software (Bio-Rad).

10 μg protein lysate of whole zebrafish (pooled from 12 animals per group) or 10 µg of podocyte cell lysate were resolved in 10 % SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Detection of protein bands was performed using horseradish peroxidase-labelled secondary antibodies and visualized using enhanced chemi-luminescence reagents (Pierce, Rockford, IL). Primary antibodies were monoclonal mouse anti-zebrafish Vegf-A antibody (R&D systems, MAB1247, Minneapolis, MN 1:1000) and anti-GAPDH (Santa Cruz, FL-335, Dallas, Texas, 1:1000). Secondary antibodies were donkey anti-mouse IgG HRP (Santa Cruz, Dallas, Texas, 1:10.000) and donkey anti-rabbit IgG HRP (Santa Cruz, Dallas, Texas 1:10.000).

The lysates of murine splenic CD4+ T cells were prepared by homogenization with CelLytic M, supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined using the Pierce BCA protein Assay kit (Thermo Fisher Scientific). The samples were run on a 4–20% Mini-PROTEIN TGX Precast Gel (Bio-Rad) electrophoresis (SDS-PAGE) and blotted on Amersham Hybond P 0.45 µm PVDF (GE Healthcare Life Sciences). Primary antibodies against CTSE at 1:2,000 dilution (NB400-152, NOVUS) and beta-Actin at 1:1,000 dilution (ab8227; abcam) in 5% bovine serum albumin in TBS supplemented with 0.1% Tween 20 (TBS-T) were incubated overnight at 4 °C. Then, the blots were washed with TBS-T and incubated with donkey anti-rabbit IgG-HRP (1:5,000; SANTA CRUZ) for 45 minutes at room temperature. After three-time washes with TBS-T, bands were visualized with enhanced chemiluminescence using the Pirece Western Blotting Substrate Plus (Thermo Fisher Scientific). Protein bands were semiquantified by densitometry using ImageQuant TL software (GE Healthcare Life Sciences).