Chemiluminescence system

RIPA buffer (Servicebio, Wuhan) was employed for total cellular protein lysis. Following ultrasonic cracking, the quantification of lysates was done by a BCA Protein Assay kit (Servicebio, Wuhan). 10% SDS‐PAGE gel was utilized for the separation of total protein, which was transferred to a PVDF membrane (Servicebio, Wuhan). Following the incubation of antibodies, a chemiluminescence system (CLINX, Shanghai) was utilized for the detection of signals. The primary antibodies (all with the same dilution ratio of 1:1000 provided by ProteinTech Group, Wuhan, China) we utilized were anti‐rabbit NCBP1(catalog no. 10349-1-AP), NCBP2(catalog no. 11950-1-AP), EIF4E(catalog no. 11149-1-AP), EIF4E3(catalog no. 17282-1-AP), EIF4E2(catalog no. 12227-1-AP), and anti‐mouse GAPDH(dilution ratio of 1:2000, Servicebio, catalog no. GB15002). The anti‐rabbit and anti‐mouse (both with a dilution ratio of 1:5000, catalog nos. GB23303 and GB25301, respectively, Wuhan, China) secondary antibodies were purchased from Servicebio. GAPDH was employed as an internal control.

The tissue distribution of the bPepT1 protein in the gastrointestinal tract of dairy cows was analyzed by Western blot analysis, as described previously [23 ]. Briefly, the epithelial tissues of rumen, omasum, duodenum, jejunum, and ileum that were isolated from seven healthy Holstein dairy cows in mid-lactation were lysed and centrifuged, and the protein concentrations were determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of proteins were electrophoresed on polyacrylamide gels and electro-transferred to a polyvinylidene difluoride membrane. The membranes were incubated overnight at 4 °C with either a rabbit anti-PepT1 antibody or an anti-β-actin antibody, and then incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. The band densities were detected using a chemiluminescence system (CLiNX Science, Shanghai, China) and quantified using Quantity One software (Bio-Rad, Hercules, CA, USA). β-actin was used as a loading control to normalize the protein bands. The band intensity of bPepT1 expressed in the rumen was set to 1 as a baseline to compare those of all other tissues.

Cells were treated with chemicals of different concentrations for 48 h, and cell lysates were obtained with RIPA buffer containing 1 mM PMSF. The protein concentration was measured with a BCA protein quantification kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated by SDS/PAGE and then blotted onto polyvinylidene difluoride membranes, which were incubated with 5% skim milk for 1 h. Immunoblotting was performed with an anti-H4R3me2s antibody (1:1000, 61988, Active Motif), anti-H3R8me2s antibody (1:1000, 13939, CST, Danvers, MA, USA), anti-cleaved PARP antibody (1:1000, 5625, CST), and anti-β-actin antibody (1:5000, 3700, CST), followed by incubation with horseradish peroxidase-linked anti-rabbit IgG (1:2000, 7074, CST) or an anti-mouse IgG secondary antibody (1:2000, 7076, CST). The membranes were imaged using a chemiluminescence system (Clinx Science Instruments, Shanghai, China).