Nf κb p65

Immunofluorescence assays were performed according to a previous study with some modifications (Mu et al., 2014 (link)). For bacterial visualization, a GFP-labeled plasmid pCA66-GFP was electrotransformated into C. sakazakii ATCC 29544. The plasmid did not affect the invasion abilities of the strain (data not shown). HBMEC were propagated on round glass coverslips within 24-well plates to 70–90% confluence and infected with GFP-labeled C. sakazakii (MOI = 100). After infection for 0, 1, 2, 3, and 4 h, the cells were washed with PBS and fixed with 4% paraformaldehyde at 4°C for 1 h. After blocking with QuickBlock^TM Blocking Buffer (Beyotime, Shanghai, China) for 20 min at room temperature, the samples were incubated with the 1:100 dilutions of primary antibodies of CD44 (Proteintech, Wuhan, China), Rab5 (Beyotime), Rab7 (Beyotime), LAMP2 (Beyotime), Cathepsin L (Proteintech), and NF-κB p65 (Proteintech) at 4°C overnight. Cy-3 labeled secondary antibodies were used at a 1:500 dilution at room temperature. Samples on the slides were washed with PBS and mounted onto microscope slides with antifade reagent with DAPI (Beyotime). The images of protein expression and distribution in HBMEC were obtained with confocal laser scanning microscopy (Revolution-XD, Andor, England).

For western blotting, cell lysis buffer for western and IP (Bestbio, Shanghai, China) was used to prepare J774A.1 cell proteins. The protein concentrations of J774A.1 were quantified using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Inc, Waltham, MA, USA). The samples of J774A.1 proteins was adjusted to a concentration of 30~50 μg perwell. After electrophoresis, protein samples were transferred to a polyvinylidene difluoride membrane that was blocked with 5% milk in TBS-T (TBS containing 0.05% Tween 20) for 1 h at room temperature. After washing the membrane with TBS-T, it was incubated with the specific antibodies against p-ERK (proteintech, Chicago, IL, USA), ERK (proteintech, USA), p-p38 (CST, Danvers, MA, USA), p38 (proteintech, Chicago, IL, USA), p-JNK (proteintech, Chicago, IL, USA), JNK (proteintech, Chicago, IL, USA), p-NF-κB p65 (CST, USA), NF-κB p65 (proteintech, Chicago, IL, USA), β-actin (proteintech, Chicago, IL, USA), or VapA (ABclonal, Wuhan, China) in dilution 1:1000 at 4 °C overnight. The membrane was washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Membrane was then washed thoroughly with TBS-T before the addition of a chemiluminescent substrate and exposure.

Tissue sections were dewaxed, rehydrated, and performed heat-mediated antigen retrieval. The sections were incubated at 4°C overnight with primary antibodies against COL2A1, ACAN, MMP13, TNFα (1 : 100, Abcam), p-PI3K (1 : 100, Cell Signaling Technology), NF-κB p65 (1 : 250, Proteintech), p16 (1 : 250, Bioss), and IL6 (1 : 200, Abcam). Then, the sections were incubated with HRP-conjugated secondary antibody, and the signal was developed with 3,3′-diaminobenzidine. The images were captured with a microscope (Leica DMI4000B, Wetzlar, Germany). Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to evaluate the dyed area and the integrated optical density (IOD), and mean density equaled the IOD value divided by area. The calculation was executed by two observers blinded to the experimental design.