Goat anti rabbit igg hrp

One hundred micrograms of denatured total proteins from liver, extracted using RIPA buffer (Merck, Germany) were separated by SDS-PAGE electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, MA, USA). Membranes were overnight incubated at 4°C with 1:1000 dilution of phospho-STAT3 (Tyr705, #9138 Cell Signaling, Beverly, MA, USA), STAT3 (#4904 Cell Signaling, Beverly, MA, USA) and β-actin (#4967 Cell Signaling, Beverly, MA, USA) primary antibodies followed by 1 h incubation at RT with horse anti-mouse IgG-HRP at 1:5000 (#7076 Cell Signaling, Beverly, MA, USA) for phospho-STAT3 antibody binding and goat anti-rabbit IgG-HRP at 1:5000 (#7074 Cell Signaling, Beverly, MA, USA) for STAT3 and β-actin antibodies binding. Chemiluminescence signal was enhanced by adding ECL (SuperSignalTM West Dura Extended Duration Substrate, Thermo Fisher Scientific, MA, USA) and quantified using ImageJ 1.8 software.

Western blotting analysis was performed as described previously [46 (link)] using primary antibodies against LCN2 (R&D Systems), ERp57, ERAP1 (Abcam; Cambridge, MA, USA), ERO1α (Novus Biologicals; Littleton, CO, USA), HO-1 (Enzo Life Sciences; Farmingdale, NY, USA), CRT, NRF2, calnexin, p47phox, β-actin (Santa Cruz Biotechnology; Dallas, TX, USA), and PDI (Cell Signaling; Danvers, MA, USA). Goat anti-rabbit-IgG-HRP (Cell Signaling), donkey anti-goat IgG (Santa Cruz Biotechnology), and rabbit anti-mouse-IgG-HRP (Calbiochem, San Diego, CA, USA) were used as secondary antibodies. The blots were quantified using an Alliance Mini 4 M (UVItec, Cambridge, UK). Western blot bands corresponding to each protein were quantified, and the intensity of each target protein was normalized to the intensity of the β-actin loading control. The normalized ratio of the control was set as 1.0 to compare target protein abundance in different sample. The normalized ratios of target proteins were used to compare target protein abundance in different samples. The normalized ratio is shown at the bottom of the blots. The normalized intensity values of three different experiments are plotted as mean ± standard deviation (SD).

K562 and KA were treated with different concentrations of DMP or at different time points. After adding the lysate (50 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.1 mM sodium orthovanadate, 50 μg/mL aprotinin, 1 mM phenylmethysulfonyl fluoride, and 10 mM Tris-HCl; pH 7.4), shock lysis was performed on the ice bath for 30 min, followed by ultrasonic nucleation on the ice bath for 30 seconds (50% strength, 2 s/4 s), centrifugation at 12000 rpm at 4°C for 15 min. After the supernatant was taken, the protein was quantified and SDS-PAGE gel electrophoresis was performed. After electrophoresis, the samples were transferred to nitrocellulocellulose membrane, followed by an immune reaction, and then incubated with c-Abl antibody (Cell Signaling Technology #2862), SRC antibody (Cell Signaling Technology #2108), β-actin (Cell Signaling Technology #8457) at room temperature for 4°C overnight. Goat anti-rabbit IgG-HRP (Cell Signaling Technology) was incubated for 2 hours, TBST was washed for 2 hours, and ECL solution was added for 1 minute. The membrane was drained and exposed in a bio-RAD chemiluminescence imager for several minutes. The results were read by ImageLab 5.2.1 software and analyzed statistically with β-actin as the internal reference.

The following primary antibodies were used in this study: anti-p44/42 MAPK (Erk1/2), rabbit polyclonal (cat. #4370S; Cell Signaling Technology, Beverly, MA) at a 1:1000 dilution; goat anti-rabbit IgG HRP (cat. #7074S; Cell Signaling Technology) at a 1:3000 dilution; anti-mouse IgG Kappa HRP, mouse monoclonal (cat. #SC-516102; Santa Cruz Biotechnology, Inc., Dallas, TX) at a 1:5000 dilution; anti-β-actin (13E5), rabbit monoclonal (cat. # 4970S; Cell Signaling Technology) at a 1:1000 dilution; anti-inhibin alpha antibody, rabbit polyclonal (cat. # ab216969; Abcam, Cambridge, UK) at a 1:100 dilution; and anti-mouse cytokeratin 8 (Krt8) rat IgG monoclonal (TROMA-I; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA ) at a 1:150 dilution.

Ferrozine, neocuproine, ascorbic acid and 3-methyladenine (3-MA) were bought from Sigma-Aldrich (USA). Ammonium acetate and 30% hydrogen peroxide (H₂O₂) solution were purchased from Aladdin (China). Antibody against LC3 (NB100-2220) and P62 (ab56416) were purchased from Novus (USA) and Abcam (USA), respectively. PathScan^® Intracellular Signaling Array Kit (7323), antibody against PARP (9542T) and Beclin-1 (3495T) and goat-anti-rabbit-IgG-HRP were all obtained from Cell Signaling Technology (USA). Goat-anti-mouse-IgG-HRP was obtained from Beyotime Biotechnology (China). Antibody against β-actin (abs-118937) was bought from Absin (China).