Rabbit anti mouse igg hrp

For the Western blots, whole-cell extracts and whole embryo extracts were prepared and resolved by 2–10% gradient SDS-PAGE. The proteins were then transferred to a PVDF membrane. The membrane was incubated with anti-GFP (Sigma, 1:1000) and anti-PCNA (Abcam, 1:5000) primary antibodies for 4 h at room temperature. A secondary antibody rabbit anti-mouse IgG HRP (abcam, 1:1000) was used for detection.

Western blotting was used to analyze the expression of osteogenic proteins during cell differentiation. The hMSCs were seeded on the test specimens at a density of 1 × 10⁴ cells/cm², and the total cell lysate was harvested after 7, 14 and 21 days of cell incubation using a radioimmunoprecipitation assay (RIPA) buffer. Then, 20 µg of the total cell lysate was separated using 10% polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes and was then blocked with 5% BSA/PBS. After blocking, the membranes were incubated with specific primary antibodies, including anti-bone sialoprotein (anti-BSP, Millipore), anti-osteopontin (anti-OPN, Abcam), anti-osteocalcin (anti-OCN, Abcam) and anti-GAPDH (Abcam); the membranes were then incubated with secondary antibodies. The secondary antibodies used for this evaluation were goat-anti-rabbit IgG-HRP (Abcam) and rabbit-anti-mouse IgG-HRP (Abcam). An ECL detection kit (Millipore) was used as the substrate to measure the specific protein expression with HRP on immunoblots, and images were obtained using a luminescence/fluorescence image system (LAS4000, Fujifilm). The relative photographic density was quantified using the Multi Gauge V2.2 software.

Dimethyl sulfoxide and fetal bovine serum (FBS) was purchased from Gibco Company. DMEM high sugar medium, trypsin, penicillin, streptomycin, phosphate buffer solution, DAPI, polyformaldehyde Mitotracker XF cell mitochondrial oxygen consumption detection kit, 1X PBS buffer, mitochondrial membrane potential detection kit, Annexin V-FI TC cell apoptosis kit, 5 × loading buffer, NC membrane, a protein phosphatase inhibitor, 4 × loading buffer, SDS-PAGE kit, 30% polyacrylamide, 1 M Tris-HCI (PH 6.8), 1.5 M Tris-HCL (PH 8.8), 10% SDS, 10% ammonium persulfate (APS), and ECL Plus hypersensitive luminescent solution were purchased from Trizol Reagent from Beijing Soleboard Technology Co., Ltd. Glutamine was purchased from Gibco Company and Sodium Pyruvate from Amresco Company. Rapamycin, anti-mTOR antibody, anti-Raptor antibody, anti-p4E-BP1 (Ser65) anti-P70s6k antibody, anti-CPT1A anti-ATP5D antibody (Mice), anti-GAPDH antibody, Shan Sheep anti-rabbit IgG/HRP, and rabbit anti-mouse IgG/HRP were purchased from Abcam.

Cells in the logarithmic growth phase were collected and lysed with the RIPA buffer containing protease and phosphatase inhibitors. Extracted proteins were loaded onto 8% SDS-PAGE at a voltage of 150 V after quantification. Thereafter, the proteins were transferred onto the PVDF membrane and incubated with the primary mouse antibodies against SCD (1 : 500, Abcam) and GADPH (1 : 1000, Abcam), respectively, overnight at 4°C. After three times being washed with TBST, the membrane was probed with the secondary antibody rabbit anti-mouse IgG (HRP) (1 : 2000, Abcam) for 1 h at room temperature. Finally, the protein bands were detected using the ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, NY, USA).

Serum obtained from cardiac bleeds on day 21 and cheek bleeds on experimental days 7 and 14 were tested for antibody response to the predicted B cell peptide epitopes used for vaccinations via peptide ELISAs. Plates were coated with 5μg/mL of target peptide using coating reagent from the Takara Peptide Coating Kit (Takara cat. #MK100). Measles peptide was utilized as a negative control, and Flag peptide was also plated as an experimental control. Plates were blocked with a blocking buffer according to the manufacturer’s protocol. Serum was plated in duplicate wells with serial dilutions, and anti-FLAG antibody was plated in the experimental control wells. Rabbit anti-mouse IgG HRP (Abcam ab97046) was utilized as a secondary antibody. TMB substrate (Thermo Fisher Scientific cat. #34028) was added, development was stopped with TMB Stop solution (BioLegend cat. #423001), and plates were read at 450 nm.