Hrp conjugated anti rabbit igg

The protein concentration of cell extracts was determined using the BCA Protein Assay Kit (Pierce, USA). Western blot analysis was performed as previously described [23 (link)]. Antibody binding was revealed using an HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma, USA). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millopore, USA).

The protocol for protein extracts from rat epididymis was designed, based on previously described procedures (36 (link)). Sample aliquots containing equal amounts of protein were separated via SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked in 5% non-fat milk for 1 h at room temperature, and incubated overnight at 4°C with rabbit anti-RANTES (Abcam, Cambridge, United Kingdom), rabbit anti-V-ATPase (GeneTex, Irvine, CA, United States), rabbit anti-iNOS (Abcam, Cambridge, United Kingdom), rabbit anti-AGTR2 (GeneTex, Irvine, CA, United States), rabbit anti-CCR1 (Abcam, Cambridge, United Kingdom), rabbit anti-CCR5 (GeneTex, Irvine, CA, United States), mouse anti-β-actin (Invitrogen, United States) and mouse anti-GAPDH (Invitrogen, United States). The membranes were then washed with TBST buffer and incubated with an appropriate secondary antibody (HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG, Sigma, St. Louis, MO, United States) for 2 h at room temperature. After extensive washing, the densities of labeled protein bands on the blots were detected using an enhanced chemiluminescence reagent (Thermo, Rockford, IL, United States) and captured using a ChemiDoc MP System (Bio-Rad, Hercules, CA, United States).

Total CYP2E1 protein levels in COS-7 cells microsomal fractions were determined by western blot analysis. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene difluoride membrane (Thermo, CA, USA). After incubation with the rabbit anti-human CYP2E1 primary antibody (Sigma-Aldrich [MO, USA], Cat. No. HPA009128) for 24 h at 4°C, the PVDF membranes were incubated with the HRP-conjugated anti-rabbit IgG (Sigma-Aldrich) for 2 h at room temperature. The expression of each variant was normalized with regard to that of the wild-type CYP2E1. Proteins were detected using the SuperSignal™ West Dura Extended Duration Substrate (Thermo Scientific Catalog No. 34075), and images were captured on a Tanon-5200Multi Chemiluminescent Imaging System (Tanon, Shanghai, China). Quantification of protein density was performed with Image J software (https://imagej.nih.gov/ij/download.html).

ELISA plates were coated with PV1, PV2, or PV3 infected or uninfected CV1cell lysates (100 μg of total protein/well) or recombinant proteins (100 ng/well) overnight at 4 °C. The plates were blocked with 3% SMP in PBS for 1 h at 37 °C. Different polyclonal sera at various dilutions (1:200 to 1:3200) in 1% SMP in PBS were added and incubated at 37 °C for 1 h, washed three times with PBST, followed by addition of HRP-conjugated anti-rabbit IgG (Sigma-Aldrich) at 1:5000 dilution in 1% SMP at 37 °C for 1 h. The plate was washed three times with PBST and once with PBS, and then developed using o-phenylene diamine (OPD) substrate. The reaction was stopped by addition of 50 μL of 3 M sulphuric acid per well and the OD was read at 490 nm. The ELISA data was analyzed using one-way analysis of variance (GraphPad Prism). The p values were calculated by comparing lysates from control CV1 cells to lysates from CV1 cells infected with PV1, PV2 or PV3, using Bonferroni's multiple comparison test.