Immobilon western chemiluminescent hrp substrate kit

For the determination of albumin and transferrin, the commercial kits “Human Albumin ELISA kit” and “Human Transferrin ELISA Kit” (both from Bethyl Laboratories, Montgomery, AL, USA) will be used, respectively. To determine the GM2 ganglioside activator protein (GM2AP), Western blotting will be performed. Briefly, 21 µL of urine from each patient per sampling time will be separated by acrylamide electrophoresis. Proteins will be transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the primary antibody against GM2AP (own production). Subsequently, they will be incubated with horseradish peroxidase-conjugated secondary antibody and chemiluminescent detection (Immobilon Western Chemiluminescent HRP Substrate kit; Millipore, Madrid, Spain) will be performed with the ChemiDoc MP imaging system (Bio-Rad, Madrid, Spain).

Total cellular proteins were extracted using Cell Lysate RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) with phenylmethanesulfonyl fluoride (PMSF, Beyotime Biotechnology, Shanghai, China) and a protease inhibitor (BestBio, Shanghai, China) at a 100:1:1 ratio. Proteins were separated by 10% SDS-PAGE and transferred to the PVDF membranes (Millipore, Burlington, MA, USA). After blocking in 5% skimmed milk, the membranes were incubated with primary antibodies against E7 (1:500; sc-6981; SANTA CRUZ, CA, USA), GAPDH (1:1000; 10494-1-AP; Proteintech, Wuhan, China), and β-actin (1:1000; ab8226; Abcam, Waltham, MA, USA). The goat anti-mouse HRP-conjugated secondary antibody (ZB-2305) and goat anti-rabbit HRP-conjugated secondary antibody (ZB-2301) were purchased from ZSGB-BIO (Beijing, China). The proteins were visualized using the Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, Burlington, MA, USA).

The 14 skeletal muscle samples were first lysed with cold T-PER Tissue Protein Extraction Reagent (Thermo, USA). Then, the protein concentration of the lysates was measured using the BCA protein kit (Beyotime, China). Thereafter, the proteins were sorted with SDS-polyacrylamide gel, and then transferred to polyvinylidene fluoride membranes (Millipore, USA). Then the membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature. Subsequently, the membranes were incubated for overnight at 4 °C using primary antibodies against CYC1, NDUFC2, NDUFS3, PC, LDHB, GPI, PKM, TPI1, PAGM2 (Proteintech, China; 1:300 dilution), α-Tublin (Abcam, 1:1000 dilution) and GAPDH (Abcam, 1:1000 dilution). The HRP-labeled anti-rabbit/mouse IgG secondary antibody (Beyotime, China; 1:2000 dilution) was incubated for 1 hour at room temperature. Finally, the protein bands were visualized using the Immobilon Western Chemiluminescent HRP substrate kit (Millipore, USA). The gray values of the protein bands were measured using ImageQuantTL software (GE, USA). Levels of the α-tubulin protein were used as internal control. T-test was conducted to analyze the statistical significance of differences between low and high- FE pigs. Significance level was set at P < 0.05.

Tissues were homogenized using metal beads in a Tissue Lyser (Qiagen). Samples were incubated on ice for 30 min and then centrifuged at 4 °C for 30 min at 14,000 rpm. The supernatant was transferred to a new tube, protein concentration was determined by the BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of lysates were resolved on SDS-PAGE gels and electrophoretically transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% (w/v) nonfat milk, and then incubated with a specific primary antibody to each target protein overnight at 4 °C. Primary antibodies were directed against a number of proteins as denoted in Supplementary Table 8. Following separate steps of washing to remove unbound antibodies, membranes were probed with a species-specific HRP-conjugated secondary antibody for 1 h. Image was detected by Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, Burlington, MA). Equivalent loading was further assessed by ponceau S staining. All blots or gels depicted in a given figure panel derive from the same experiment in which samples were processed in parallel.

Whole cell protein was extracted with Radio Immunoblot Precipitation Assay buffer (RIPA) and conventionally treated for specific immunodetection by western blot technique [52 (link)] of mouse Rad51c protein in cells treated with miR-222 or transfection control. We used an anti-Rad51c polyclonal antibody that recognizes RELVGYPLSPAVRGKGKLVAAGFQTAED, corresponding to N terminal amino acids 3–28 of Mouse Rad51C (Cat. ab95201, Abcam; Cambs., UK) and detected ß-tubulin protein as an endogenous control for the experiments with a mouse anti-ß-tubulin monoclonal antibody (Cat. 322600, Invitrogen, Camarillo, CA, USA). Horseradish Peroxidase (HRP)-coupled goat anti-rabbit IgG (immunoglobulin G) monoclonal (Cat. 816129, Invitrogen, Camarillo, CA, USA) and goat anti-mouse IgG monoclonal (Cat. 626520, Invitrogen, Camarillo, CA, USA) secondary antibodies were utilized for Rad51c and ß-tubulin radiographical detection, respectively, with the Immobilon Western Chemiluminescent HRP Substrate kit (Millipore; MA, USA). Protein quantification was performed with Kodak 1D Image Analysis v3.5 software and expressed as band intensity at optical densities (D.O.).

Western Blots were performed according to the protocols described previously [22 (link)]. The Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, MA, USA) was used to detect the results. Primary Antibody are ERK1/2 (AM076, 1:500; Beyotime, Nanjing, China), and phospho-ERK1/2 (AM071, 1:500; Beyotime, Nanjing, China).

Total protein extracts were prepared from cell samples (macrophages harvested at 12 and 24 hpi) using commercial kits (Pierce, USA). Protein concentrations were determined by bicinchoninic acid (BCA) assay and the samples were stored at −70°C until analyzed.
Cells were lysed in RIPA lysis buffer (1% Triton X-100, 0.25% deoxycholate (DOC), 0.05% SDS, 50 mM Tris pH8.0, 150 mM NaCl and 50 mM NaF) containing proteinase inhibitor cocktails (Roche Applied Science, Mannheim, Germany) for 30 min at 4°C. The concentration of protein in the lysates was assessed with the BCA protein assay reagent (Pierce Biotechnology, IL, USA). Cell lysates were analyzed by SDS-PAGE, and proteins were transferred to PVDF membranes (Millipore, MA, USA). Membranes were subsequently incubated with a primary antibody of RIG-I, TLR-3, TLR-7, MAVS and MyD88 (Cell Signaling Technology) followed by horseradish peroxidase (HRP)-conjugated secondary antibody (Bethyl Laboratories, Inc., TX, USA). Signals were detected using an Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, Shanghai, China).