Xrs system

RIPA reagent was used to lysate cells and leach total protein. The same amounts of protein samples were separated in SDS-PAGE and then transferred to the PVDF membrane. The membrane was soaked in 5% skim milk at room temperature for 2 h. Rabbit anti-human JAG1 monoclonal antibody (1:1000, # ab85763, Abcam, USA) or GAPDH monoclonal antibody (1:2000, # ab9485, Abcam, USA) was incubated at 4 °C overnight. Finally, the membrane was incubated with a secondary antibody (1:5000, #ab6721, Abcam, USA). The proteins were exposed and photographed in a ChemiDoc XRS⁺ system.

To explore if M. hyopneumoniae EF-Tu could bind to fibronectin, the protein-protein interactions method far-Western blot (far-WB) was performed. Total 20 μg recombinant proteins, including rEF-Tu and a negative control MRP-D2, which was previously verified not to bind to fibronectin (Li et al., 2017 (link)) were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Li et al., 2015 (link)). After blocked with 5% (w/v) skimmed milk, the membrane was incubated with 5 μg/mL fibroenctin (Sigma), followed by incubation with rabbit anti-fibronectin antibody (Boster; 1 μg/mL) as the primary antibody, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Boster; 1:5,000 dilution) as the secondary antibody. Finally, the membrane was developed with ECL substrate using a ChemiDoc XRS+ system. Polyclonal antibody against rEF-Tu was used as the positive control with the same process.

A nitrocellulose membrane was dotted with purified α-enolase at each concentration and incubated at 4 °C for 15 min. After washing with TBST, the membrane was blocked in a 5% BSA solution for 2 h. Subsequently, the indicated compound or DMSO was applied to the membrane in the 5% BSA solution for 4 h at room temperature. After washing with TBST, the membrane was incubated with a secondary antibody (anti-biotin-HRP) in 5% BSA at room temperature for 2 h. Subsequently, the membrane was developed using Clarity Western ECL Substrate and visualized using the ChemiDoc XRS+ system.

The purified HAP103 Ab was analyzed via SDS-PAGE gels with samples loaded with reducing or nonreducing buffer. After staining with Coomassie blue, the gel was washed with distaining solution overnight, and the results were recorded with a ChemiDoc XRS system.
HCC cells were lysed in RIPA buffer. The samples were quantified by a BCA kit, resolved by SDS-PAGE, transferred onto a PVDF membrane, blotted with mouse anti-PKM2 (1:1000), anti-β-actin (1:4000) and anti-Hif1a (1:500) PcAbs, and finally detected with HRP-labeled secondary Abs.

In this study, the Proteome Profiler Human Cytokine Array Kit (ARY005B, RD, USA) was used to detect the expression of cytokines in H. pylori-infected macrophage culture medium. The assay was performed according to the manufacturer’s instructions. In brief, the membranes were blocked and then incubated with antibody-sample mixture overnight at 4℃. The membranes were incubated with secondary antibody. The membranes were imaged and quantified using the ChemiDoc XRS + system. Semi-quantitative analysis was performed by grayscale statistics.