Ab213676

RAW 264.7 macrophages were treated with or without YfeA (20 µg/mL) for 12 h, the total cellular protein was extracted using a Protein Extracting Kit (Solarbio, Beijing, China). Aliquots corresponding to 50 µg of each sample were analyzed using Western blotting (WB). To analyze the YfeA-induced signal transduction residue phosphorylation, the primary monoclonal antibodies, including Erk1/2 (rabbit, 42/44 kDa; 1:2000; Abcam ab184699), p-Erk1/2 (rabbit, p-ERK1: Thr202/Tyr204, p-ERK2: Thr185/Tyr187; 42/44 kDa; 1:2000; Abcam ab278538), p38-MAPK (rabbit, 41 kDa; 1:1000; Abcam ab31828), p-p38-MAPK(rabbit, Tyr182; 41 kDa; 1:500; Abcam ab47363), JNK (rabbit, 48 kDa; 1:1000; Abcam ab179461), p-JNK (rabbit, Tyr185/223; 48 kDa; 1:10,000 Abcam ab76572), p65 (NF-κB; rabbit, 60 kDa; 1:2000; Abcam ab16502), p-p65 (NF-κB; rabbit, Ser536; 60 kDa; 1:5000; Abcam ab86299), TLR2 (rabbit, 89 kDa; 1:500; Abcam ab213676), and TLR4 (rabbit, 89 kDa; 1:500; Abcam ab13556) were applied at this stage.

The total proteins of intestinal tissues were extracted by RIPA protein lysis buffer (Invitrogen; USA), and the concentration and purity of proteins were measured by a BCA protein kit (Pierce; USA). Then, 40 μg of protein from each sample was separated by SDS–PAGE, transferred to PVDF membranes by the electric transfer method and blocked with skim milk at room temperature for 1 h. After incubation with primary and secondary antibodies, the protein bands were visualized by enhanced chemiluminescence (Pierce; USA). The primary antibodies were diluted at 1:1000 as follows: anti-ZO-1 (ab96587; Abcam; UK), anti-Occludin (ab216327; Abcam; UK), anti-TLR2 (ab213676; Abcam; UK), anti-Tollip (ab187198; Abcam; UK), anti-MyD88 (ab219413; Abcam; UK), anti-IRAK1 (ab238; Abcam; UK), anti-p-IRAK1 (ab218130; Abcam; UK), anti-p65 (ab16502; Abcam; UK), anti-p-p65 (ab76302; Abcam; UK), anti-MLCK (ab232949; Abcam; UK), and anti-GAPDH (ab8245; Abcam; UK). The grayscale value analysis was performed by using ImageJ software (version 1.52a; National Institutes of Health), and GAPDH was used as the loading control.

The BD Accuri C6 Software was used to investigate TLR4 and TLR2 expression on the surface of hDPSCs stimulated by LPS (1 μg/mL) from E. coli or P. gingivalis. Cells were collected, washed with PBS, counted, and then resuspended in the staining buffer. Cells were incubated with the anti-TLR4 antibody (Abcam) (ab13556) or anti-TLR2 antibody (Abcam) (ab213676) for 1 h at 4 °C. The secondary antibody diluted to 1/2000 was added for another 30 min in the dark (Abcam) (ab150079). The isotype control antibody (Abcam) (ab37415) was used under the same conditions. Data analysis was performed using the FlowJo 10.6.2 software.

Uric acid (batch number BCBH0278V) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, United States). Colchicine (batch number 20190901) was purchased from Xishuangbanna Pharmaceutical Co., Ltd. Rat IL-1β (batch number MM0047R1) and TNF-α (batch number MM0180R1) ELISA kits were purchased from Meimian Co., Ltd. (Yancheng, China). Mouse TNF-α (batch number CSB-E04741m) ELISA kit was purchased from Cusabio Co., Ltd. (Wuhan, China). Primary antibodies against TLR2 (ab213676), TLR4 (ab217274), MyD88 (ab2064), NF-κB (ab76302), NLRP3 (ab214185), caspase-1 (ab138483), and IL-1β (ab9772) were purchased from Abcam Inc. (Cambridge, MA, United States). RAW264.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Western blot analyses were performed as previously described32 (link). Briefly, cerebral tissue samples were collected, homogenized, and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% polyacrylamide gels. A BCA Protein Assay Kit (Beyotime) was used to measure protein concentrations with the bicinchoninic acid method. After separation, protein samples were transferred onto Immobilon nitrocellulose membranes. The membranes were blocked with 5% nonfat milk at room temperature for 1 h. The membranes were then incubated with the following primary antibodies overnight at 4°C: rabbit anti-β-actin (1:1,000, rabbit polyclonal, Abcam, ab8227), rabbit anti-Cleaved-Caspase-3 (1:5,000, Abcam, ab214430), rabbit anti-TLR2 (1:1,000, ab213676), and rabbit anti-TLR4 (1:1,000, rabbit polyclonal, Abcam, ab13556). After washing the membranes with Tris buffered saline tween (TBST) three times, horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (1:5,000) were applied, and the membranes were incubated with the secondary antibodies at room temperature for 1.5 h. The protein bands were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, United States of America) and quantified with ImageJ software.

The following antibodies were used in the study: rabbit anti-human TLR2 (ab213676 at dilution 1:200), mouse anti-human TLR4 (ab22048 at dilution 1:100), rabbit anti-human TLR7 (ab124928 at dilution 1:100), mouse anti-human TLR9 (ab134368 at dilution 1:200), rabbit anti-human RAGE (ab3611 at dilution 1:100), rabbit anti-human HMGB1 (ab79823 at dilution 1:400), and rabbit anti-human NF-κB p65 (ab16502 at dilution 1:1000) (all from Abcam, UK).