antibodies against paf receptor (sc-8744)  (Santa Cruz Biotechnology)

Journal: International Journal of Molecular Sciences

Article Title: Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells

doi: 10.3390/ijms25063084

Figure Lengend Snippet: PAFR expression in CE cells. ( A ) Schematic diagram showing mouse peripheral eye anatomy and indicating the retina (RE), iris (IR), lens, and the ciliary epithelium (CE) divided into the inner non-pigmented epithelium (NPE) and the outer pigmented epithelium (PE). The PE can form neurospheres (NS) after growth factor stimulation. ( B ) Immunohistochemistry analysis of eyes’ sections showing cells positive for PAFR (green) and nuclei (DAPI) in both PE and NPE cells, as indicated by the arrowheads in the detailed image in “A” ( n = 3). ( C ) The detailed image from figure ( B ) indicates PAFR expression in the cell membrane/cytoplasm (regular arrows) and in the nuclei (arrowheads). The white bar represents 25 μm. ( D ) PAFR and DAPI confocal fluorescence profile analysis demonstrated fluorescence expression patterns. Both fluorescence peaks colocalization (arrowhead) suggest that PAFR is present in the nuclei. The absence of colocalization peaks (regular arrow) indicates PAFR located outside the cell nuclei (green only). ( E – G ) Dissociated PE and NPE transcriptional expression of PAF receptor Pafr ( E ), PAF—synthesis enzyme Lpcat and ( F ) PAF—degradation enzyme Pafah ( G ). ( H ) PAF-related enzyme ratio expression, indicating higher levels of Lpcat than Pafah in NPE in comparison to PE. Results are given as mean ± S.E.M, * p < 0.05; ** p < 0.01; A.U. = arbitrary unit.

Article Snippet: To evaluate the influence of PAF signaling, PE cells were treated with either 100 nM of agonist cPAF (1-hexadecyl-2-N-methylcarbamyl glycerophosphocholine—carbamyl-PAF) or 10 μM of PAF receptor antagonist PCA4248 (Tocris Bioscience, Bristol, UK) every 48 h during neurosphere formation.

Techniques: Expressing, Immunohistochemistry, Membrane, Fluorescence, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells

doi: 10.3390/ijms25063084

Figure Lengend Snippet: Bioinformatic analysis on gene expression of zebrafish-derived retinal stem cells (RSC). ( A ) The quantitative transcriptional analysis indicated higher PAFR (ptafr) expression in quiescent adult progenitor cells in the CMZ ( n = 3) compared to activated developing RSC ( n = 4). ( B , C ) The quantitative transcriptional analysis indicated increased PAF-synthesis enzyme lpcat ( B ) and decreased PAF-degradation enzyme pafah ( C ) in CMZ cells. ( D ) The CMZ presented a higher transcriptional ratio of pafah/lpcat2, suggesting enhanced PAF production on differentiated tissue. CMZ = Ciliary marginal zone. RSC = Retinal stem cells. Results are given as mean ± SEM of gene counts after log2 normalization. * p < 0.05, ** p < 0.01. Retrieved scRNA-seq datasets from Xu et al., 2020 .

Article Snippet: To evaluate the influence of PAF signaling, PE cells were treated with either 100 nM of agonist cPAF (1-hexadecyl-2-N-methylcarbamyl glycerophosphocholine—carbamyl-PAF) or 10 μM of PAF receptor antagonist PCA4248 (Tocris Bioscience, Bristol, UK) every 48 h during neurosphere formation.

Techniques: Gene Expression, Derivative Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells

doi: 10.3390/ijms25063084

Figure Lengend Snippet: Progenitor/pluripotent markers expression on the pigmented epithelium (PE) of PAFR null mice (PAFR−/−). ( A , B ) The expression of CE differentiation (Palmd) and cell proliferation (Ki67) transcripts. ( C ) The total protein expression of cell cycle marker Cyclin A2. ( D ) The transcriptional expression of neural marker NeuroD1. ( E , F ) Retinal progenitor marker Pax6 transcripts and protein expression. ( G – I ) Pluripotent markers Nanog, Sox2, and Oct4 expression. Results are given as mean ± SEM. ( n = 4). * p < 0.05, *** p < 0.0005; **** p < 0.0001; ns = non-significant. A.U. = arbitrary unit.

Article Snippet: To evaluate the influence of PAF signaling, PE cells were treated with either 100 nM of agonist cPAF (1-hexadecyl-2-N-methylcarbamyl glycerophosphocholine—carbamyl-PAF) or 10 μM of PAF receptor antagonist PCA4248 (Tocris Bioscience, Bristol, UK) every 48 h during neurosphere formation.

Techniques: Expressing, Marker

Journal: International Journal of Molecular Sciences

Article Title: Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells

doi: 10.3390/ijms25063084

Figure Lengend Snippet: PAF receptor and PAF-related enzyme expression analysis on PE cells and neurospheres. ( A ) Immunohistochemistry analysis of neurospheres for PAFR (green), cell nuclei (DAPI, blue), and the merged image ( n = 3). ( B ) Detailed image from the figure ( A ) indicating PAFR expression in the cell membrane/cytoplasm (regular arrows) and in the nuclei (arrowheads). The white bar represents 25 μm. ( C ) Confocal fluorescence profile analysis indicating lower rates of colocalization peaks, suggesting that PAFR is located outside the cell nuclei (green only). ( D – G ) PAFR, PAF-related enzymes, and LPCAT2/PAFAH ratio mRNA expression in the pigmented epithelium (EP) and in PE-derived neurospheres (NS). Data are given as mean ± SEM of the comparative delta-CT ( n = 4). ns = non-significant. A.U. = arbitrary unit.

Article Snippet: To evaluate the influence of PAF signaling, PE cells were treated with either 100 nM of agonist cPAF (1-hexadecyl-2-N-methylcarbamyl glycerophosphocholine—carbamyl-PAF) or 10 μM of PAF receptor antagonist PCA4248 (Tocris Bioscience, Bristol, UK) every 48 h during neurosphere formation.

Techniques: Expressing, Immunohistochemistry, Membrane, Fluorescence, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells

doi: 10.3390/ijms25063084

Figure Lengend Snippet: PAFR agonist and antagonist effect on pigmented epithelium (PE) neurosphere formation. Optical microscopy analysis after 7 days of treatment with ( A ) control vehicle (1 μM DMSO), ( B ) 100 nM of PAFR agonist (cPAF), or ( C ) 10 μM of PAFR antagonist (PCA4248). Black arrowheads indicate neurospheres and the scale bar represents 25 μm. ( D ) Neurosphere counting analysis. ( E ) Neurosphere size measured in pixel density. ( F ) Transcriptional expression of proliferation marker ki67. ( G ) Transcriptional expression of the ciliary epithelium marker Palmd. Results are given as mean ± SEM, * p < 0.05, ** p < 0.01, **** p < 0.0001, ns = non-significant ( n = 4). A.U. = arbitrary unit.

Article Snippet: To evaluate the influence of PAF signaling, PE cells were treated with either 100 nM of agonist cPAF (1-hexadecyl-2-N-methylcarbamyl glycerophosphocholine—carbamyl-PAF) or 10 μM of PAF receptor antagonist PCA4248 (Tocris Bioscience, Bristol, UK) every 48 h during neurosphere formation.

Techniques: Microscopy, Control, Expressing, Marker

Journal: International Journal of Molecular Sciences

Article Title: Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells

doi: 10.3390/ijms25063084

Figure Lengend Snippet: PAFR agonist and antagonist’s effect on progenitor/stem cell markers. ( A – D ) mRNA expression of progenitor and pluripotent markers after 7 days of treatment with control vehicle (1 μM DMSO), 100 nM of PAFR agonist (cPAF), or 10 μM of PAFR antagonist (PCA4248). The white bar represents 25 µm ( n = 4). ( E ) Immunohistochemistry analysis on nestin expression (neural progenitor marker) and cell nucleus (DAPI) ( n = 3). ( F ) Mean fluorescence intensity (MFI) analysis for NESTIN. Results are given as the mean ± SEM of comparative delta-CT. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = non-significant. A.U. = arbitrary unit. Scale bar = 25 µm.

Article Snippet: To evaluate the influence of PAF signaling, PE cells were treated with either 100 nM of agonist cPAF (1-hexadecyl-2-N-methylcarbamyl glycerophosphocholine—carbamyl-PAF) or 10 μM of PAF receptor antagonist PCA4248 (Tocris Bioscience, Bristol, UK) every 48 h during neurosphere formation.

Techniques: Expressing, Control, Immunohistochemistry, Marker, Fluorescence

Journal: Vascular Health and Risk Management

Article Title: Relating patenting and peer-review publications: an extended perspective on the vascular health and risk management literature

doi: 10.2147/VHRM.S14454

Figure Lengend Snippet: International patent applications concerning atherosclerosis, July 2010 to January 2011 (excluding antihyperlipidemic, antidiabetic, and nonsepecific anti-inflammatory drugs)

Article Snippet: WO/2011/005608 , Merck Sharpe and Dohme (US) , Cyclohexyl sulfonamide platelet-activating factor (PAF) receptor antagonists , January 13, 2011 , None.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: ROBO4 deletion ameliorates PAF-mediated skin inflammation via regulating the mRNA translation efficiency of LPCAT1/LPCAT2 and the expression of PAF receptor

doi: 10.7150/ijbs.35797

Figure Lengend Snippet: ROBO4 deletion suppressed PAF production and expression of PAF receptor. A,B, PAF levels were increased in the skin of the pups nursed by ROBO4 KO mothers compared to that nursed by WT mothers(Control). Tissue lipids were analyzed by MRM LC-MS/MS to quantify PAF-C16 (left)(A) and PAF-C18 (right)(B) levels (n=3). The results were normalized to d4-PAF-C16 or d4-PAF-C18 internal control, respectively. P,postnatal day. C,D, The mRNAs expression of PAF synthases,LPCAT1(C) and LPCAT2(D) were unaltered in the ROBO4 KO BM macrophage(BMM,n=4), and Spleen macrophage(SM,n=4) compared to WT macrophage(n=4); 1×10 6 macrophages were differentiated from 1.5×10 6 bone marrow cells or 5×10 6 splenocytes of ROBO4 KO mice or WT control mice (2 month old). E,F, the proteins expression of LPCAT1(E,F low panel) and LPCAT2(E,F up panel) in both spleen(E) and bone marrow (F)macrophage were observably decreased in ROBO4 KO macrophage. GAPDH was used as a loading control. The macrophage differentiated manner was the same as in C.D. G, Serum level of PAFAH was elevated in the pups nursed by ROBO4 KO mothers compared to the pups nursed by WT control mothers (n=8, P21). H,I, The protein (H) and mRNA(I) expression of PAFR diminished in ROBO KO but elevated in VLDLR KO macrophage. WT, wild type; RB,ROBO4 deletion; VL,VLDLR deletion. The macrophage differentiated manner was the same as in C.D. GAPDH was used as a loading control. J,K, The protein and mRNA expression were also down regulated in ROBO4 deletion skin tissues compared to WT(wild type). Skin tissues from ROBO4 deletion or WT mice( six mice /each genotype, randomly classified into two groups) were mixed for the total protein extraction and detected by PAFR specific antibody(J);the mRNA expression of PAFR in skin tissues was tested in ROBO4 deletion or WT mice at postnatal 19(P19),postnatal 21(P21) and postnatal 27(P27) (K). Statistical analyses were performed with Student's t-Test and are shown as mean ± standard deviation;*, p<0.05; Experiment repeated at least three times. BMM, bone marrow macrophage; SM, spleen macrophage; ROBO4 KO, ROBO4-/-.

Article Snippet: Anti-PAF receptor antibody was purchased from Abcam, USA.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Protein Extraction, Standard Deviation

Journal: PLoS ONE

Article Title: Platelet Activating Factor Blocks Interkinetic Nuclear Migration in Retinal Progenitors through an Arrest of the Cell Cycle at the S/G2 Transition

doi: 10.1371/journal.pone.0016058

Figure Lengend Snippet: A: Washed rabbit platelets were treated with two independent samples of retina-derived PAF- like . Positive controls were commercial PAF (10 nM) and thrombin (50 nM), WEB2086 was used at 1 µM, and silica scraped from remote areas of the TLC plate was used as negative control. Note that the PAF receptor antagonist blocked platelet aggregation induced by retina-derived PAF- like , but not by thrombin; B: PAF- like subject to reverse phase HPLC eluted as a single peak (continuous line), coincident with pure commercial PAF (dotted line); C: Western blot of PAFR among a total protein extract from P2 rat retina shows a single band. D–F: Immunohistochemical detection of PAF receptor in transverse sections of newborn rat eyes. A negative control without primary antibody is shown in F. Arrows in D show labeling consistent with a membrane receptor in various retinal layers; Arrows in E, F show optic nerve axons. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; NBL = neuroblastic layer. Scale bars in D = 50 µm; E, F = 100 µm.

Article Snippet: PAF, C-PAF, lyso-PAF, PD98059 and LY294002 were from Calbiochem; WEB2086 was a gift from Boehringer Ingelheim; SB218078 was from Glaxo-Smith Kline; Forskolin, cheleritrin chloride, SB239063, caffeine, 5-Bromo-2-deoxyuridine (BrdU), DNAse (D4513), Hepes and glutamine from Sigma; Basal Medium of Eagle, gentamicin from Gibco BRL; antibody against BrdU (RPN 202) from Amersham Pharmacia, antibody against phospho-histone H3 (9701) from Cell Signaling; protein A/G plus agarose and antibodies against PAF receptor (sc-8744) and CHK1 were from Santa Cruz Biotechnology; and antibody against phospho-histone H2AX Ser 139 (clone JBW301) from Upstate Biotechnology.

Techniques: Derivative Assay, Negative Control, Western Blot, Immunohistochemical staining, Labeling

Journal: PLoS ONE

Article Title: Platelet Activating Factor Blocks Interkinetic Nuclear Migration in Retinal Progenitors through an Arrest of the Cell Cycle at the S/G2 Transition

doi: 10.1371/journal.pone.0016058

Figure Lengend Snippet: P2 retinal explants, pre-labeled with BrdU, were treated with 0.3 nM PAF for 3 h in the presence of various PAF receptor (PAFR) antagonists or signaling inhibitors. A: Both a PAFR-ligand inactive PAF metabolite (Lyso-PAF 10 nM, left) and a PAFR antagonist (WEB2086 10 nM, right) prevented the effect of PAF; B: Deletion of PAFR abrogates the effect of PAF upon interkinetic nuclear migration; C–E: Antagonists of both Erk (C) and p38 (D) MAP kinases prevented the effect of PAF, whereas an inhibitor of protein kinase C (E) had no effect. Data are means ± S.E.M., n = 4 in duplicate. ** = p<0.001.

Article Snippet: PAF, C-PAF, lyso-PAF, PD98059 and LY294002 were from Calbiochem; WEB2086 was a gift from Boehringer Ingelheim; SB218078 was from Glaxo-Smith Kline; Forskolin, cheleritrin chloride, SB239063, caffeine, 5-Bromo-2-deoxyuridine (BrdU), DNAse (D4513), Hepes and glutamine from Sigma; Basal Medium of Eagle, gentamicin from Gibco BRL; antibody against BrdU (RPN 202) from Amersham Pharmacia, antibody against phospho-histone H3 (9701) from Cell Signaling; protein A/G plus agarose and antibodies against PAF receptor (sc-8744) and CHK1 were from Santa Cruz Biotechnology; and antibody against phospho-histone H2AX Ser 139 (clone JBW301) from Upstate Biotechnology.

Techniques: Labeling, Migration

Journal: PLoS ONE

Article Title: Platelet Activating Factor Blocks Interkinetic Nuclear Migration in Retinal Progenitors through an Arrest of the Cell Cycle at the S/G2 Transition

doi: 10.1371/journal.pone.0016058

Figure Lengend Snippet: A–C: Treatment with PAF does not block DNA replication. Tritiated thymidine was equally incorporated in BrdU-labeled control and PAF-treated explants (A), irrespective of blockade of interkinetic nuclear migration (B and C are controls from the same batch of explants used for the 3 H-TDR measurements); ** = p<0.001. D: Arrested nuclei do not progress along the cell cycle. Explants from the retina of animals pre-injected with BrdU were maintained either in control medium or treated with 0.3 nM PAF for various intervals, and retinal sections were stained with antibodies to BrdU (green in the inset) and to phospho-histone H3 (red). Despite the blockade of nuclear migration, no immunolabeling was detected among nuclei arrested within the inner 2/3 of the neuroblastic layer (inset), and all pH3-labeled nuclei were located within the outer (apical) 1/3, albeit with a distinctive reduction in PAF-treated explants. Data are means ± S.E.M., n = 4 in duplicate; ** = p<0.001 vs. control at same time in vitro .

Article Snippet: PAF, C-PAF, lyso-PAF, PD98059 and LY294002 were from Calbiochem; WEB2086 was a gift from Boehringer Ingelheim; SB218078 was from Glaxo-Smith Kline; Forskolin, cheleritrin chloride, SB239063, caffeine, 5-Bromo-2-deoxyuridine (BrdU), DNAse (D4513), Hepes and glutamine from Sigma; Basal Medium of Eagle, gentamicin from Gibco BRL; antibody against BrdU (RPN 202) from Amersham Pharmacia, antibody against phospho-histone H3 (9701) from Cell Signaling; protein A/G plus agarose and antibodies against PAF receptor (sc-8744) and CHK1 were from Santa Cruz Biotechnology; and antibody against phospho-histone H2AX Ser 139 (clone JBW301) from Upstate Biotechnology.

Techniques: Blocking Assay, Labeling, Migration, Injection, Staining, Immunolabeling, In Vitro

Journal: PLoS ONE

Article Title: Platelet Activating Factor Blocks Interkinetic Nuclear Migration in Retinal Progenitors through an Arrest of the Cell Cycle at the S/G2 Transition

doi: 10.1371/journal.pone.0016058

Figure Lengend Snippet: A, B: Analysis of cyclin B1 content in BrdU-labeled cells. A: Cytofluorograms gated on BrdU-labeled cells dissociated from either control or PAF-treated retinal explants shows displacement of PAF-treated cells towards lower levels of cyclin B1; BKG = controls without primary cyclin B1 antibody; B: Mean and S.E.M. of the median cyclin B1 fluorescent intensity in BrdU-labeled cells, averaged among 4 independent experiments. Data were normalized to the respective controls in each experiment; * = p<0.01. C: Treatment of retinal explants with 0.3 nM PAF for 3 h induces an increase in the activity of Chk1, as shown by increased phosphorylation of a target cdc25C peptide (n = 2 with identical results) D: SB218078, a Chk1 inhibitor, blocks the effect of PAF (Data are means ± S.E.M., from n = 5 independent experiments in duplicate); ** = p<0.001.

Article Snippet: PAF, C-PAF, lyso-PAF, PD98059 and LY294002 were from Calbiochem; WEB2086 was a gift from Boehringer Ingelheim; SB218078 was from Glaxo-Smith Kline; Forskolin, cheleritrin chloride, SB239063, caffeine, 5-Bromo-2-deoxyuridine (BrdU), DNAse (D4513), Hepes and glutamine from Sigma; Basal Medium of Eagle, gentamicin from Gibco BRL; antibody against BrdU (RPN 202) from Amersham Pharmacia, antibody against phospho-histone H3 (9701) from Cell Signaling; protein A/G plus agarose and antibodies against PAF receptor (sc-8744) and CHK1 were from Santa Cruz Biotechnology; and antibody against phospho-histone H2AX Ser 139 (clone JBW301) from Upstate Biotechnology.

Techniques: Labeling, Activity Assay

Journal: Veterinary Research

Article Title: Anti-inflammatory effects of the prostaglandin D 2 /prostaglandin DP1 receptor and lipocalin-type prostaglandin D 2 synthase/prostaglandin D 2 pathways in bacteria-induced bovine endometrial tissue

doi: 10.1186/s13567-022-01100-6

Figure Lengend Snippet: Primer sequences for qPCR

Article Snippet: The following chemicals, reagents, and other materials were used in this study: foetal bovine serum (ExCellBiology, Inc., Shanghai, China); Dulbecco’s modified Eagle medium (DMEM)/F-12, penicillin, and streptomycin (Gibco, Grand Island, NY, USA); amphotericin B (GENERAY, Shanghai, China); bovine interleukin (IL)-6 enzyme-linked immunosorbent assay (ELISA) reagent kit (DY8190) and bovine tumour necrosis factor (TNF)-α duo set (DY2279; R&D Systems, Minneapolis, MN, USA); bovine IL-1β ELISA reagent kit (ESS0027; Kingfisher Biotech, St. Paul, MN, USA); Six-well culture plates (Corning, Inc., Corning, NY, USA); T-PER tissue protein extraction reagent, Halt Protease Inhibitor, Pierce BCA Protein Assay Kit, and prestained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA); sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (TAKARA, Shiga, Japan); centrifugal filter units (Millipore, Billerica, MA, USA); SDS-PAGE kit (Solarbio, Beijing, China); 10X Tris/Glycine buffer (Bio-Rad Laboratories, Hercules, CA, USA); transfer membranes (Millipore); Starting Block T20 (TBS) Blocking Buffer (Thermo Fisher Scientific); Halt Protease Inhibitor (Thermo Fisher Scientific); antibody dilution reagent (Beyotime, Shanghai, China); anti-matrix metalloproteinase (MMP)-2 antibody (Abcam, ab97779, Cambridge, UK); anti-platelet-activating factor receptor (PAFR) antibody (Biorbyt, orb11225, Cambridge, UK); and anti-high mobility group box (HMGB)-1 antibody (Novus Bio, NB100-2322, Littleton, CO, USA); Goat anti-rabbit IgG horseradish peroxidase-linked antibodies and goat anti-mouse IgG horseradish peroxidase-linked antibodies (Cell Signaling Technology, 7074 and 7076, Danvers, MA, USA); goat anti-rabbit IgG H&L antibodies (Alexa Fluor 647) preadsorbed (Abcam, ab150083, Cambridge, UK); AxyPrep Multisource Total mRNA Miniprep Kit (Axygen Scientific, Union City, CA, USA); Primer Script RT Master Mix (Takara); FastStart Universal SYBR Green Master (Rox; Roche, Basel, Switzerland); Luria Bertani broth (Oxoid, Hampshire, UK); Mueller-Hinton II cation adjusted broth (MH broth; BD Biosciences, Franklin Lakes, NJ, USA); and optimal cutting temperature compound (Sakura, Torrance, CA, USA).

Techniques: Sequencing, Concentration Assay

Journal: Veterinary Research

Article Title: Anti-inflammatory effects of the prostaglandin D 2 /prostaglandin DP1 receptor and lipocalin-type prostaglandin D 2 synthase/prostaglandin D 2 pathways in bacteria-induced bovine endometrial tissue

doi: 10.1186/s13567-022-01100-6

Figure Lengend Snippet: PGD 2 -mediated regulation of PAFR and MMP-2 via the L-PGDS/PGD 2 and PGD 2 /DP1 pathways in E. coli - and S. aureus -infected bovine endometrial explants. Immunofluorescence staining, Western blotting, and qPCR results for PAFR ( A ) and MMP-2 ( B ). Data are given as the means ± SEMs. The significance of differences between results was determined by one-way ANOVA, followed by the Dunnett’s test to control for the number of comparisons ( n = 3). Different letters indicate significantly different means ( P < 0.05).

Article Snippet: The following chemicals, reagents, and other materials were used in this study: foetal bovine serum (ExCellBiology, Inc., Shanghai, China); Dulbecco’s modified Eagle medium (DMEM)/F-12, penicillin, and streptomycin (Gibco, Grand Island, NY, USA); amphotericin B (GENERAY, Shanghai, China); bovine interleukin (IL)-6 enzyme-linked immunosorbent assay (ELISA) reagent kit (DY8190) and bovine tumour necrosis factor (TNF)-α duo set (DY2279; R&D Systems, Minneapolis, MN, USA); bovine IL-1β ELISA reagent kit (ESS0027; Kingfisher Biotech, St. Paul, MN, USA); Six-well culture plates (Corning, Inc., Corning, NY, USA); T-PER tissue protein extraction reagent, Halt Protease Inhibitor, Pierce BCA Protein Assay Kit, and prestained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA); sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (TAKARA, Shiga, Japan); centrifugal filter units (Millipore, Billerica, MA, USA); SDS-PAGE kit (Solarbio, Beijing, China); 10X Tris/Glycine buffer (Bio-Rad Laboratories, Hercules, CA, USA); transfer membranes (Millipore); Starting Block T20 (TBS) Blocking Buffer (Thermo Fisher Scientific); Halt Protease Inhibitor (Thermo Fisher Scientific); antibody dilution reagent (Beyotime, Shanghai, China); anti-matrix metalloproteinase (MMP)-2 antibody (Abcam, ab97779, Cambridge, UK); anti-platelet-activating factor receptor (PAFR) antibody (Biorbyt, orb11225, Cambridge, UK); and anti-high mobility group box (HMGB)-1 antibody (Novus Bio, NB100-2322, Littleton, CO, USA); Goat anti-rabbit IgG horseradish peroxidase-linked antibodies and goat anti-mouse IgG horseradish peroxidase-linked antibodies (Cell Signaling Technology, 7074 and 7076, Danvers, MA, USA); goat anti-rabbit IgG H&L antibodies (Alexa Fluor 647) preadsorbed (Abcam, ab150083, Cambridge, UK); AxyPrep Multisource Total mRNA Miniprep Kit (Axygen Scientific, Union City, CA, USA); Primer Script RT Master Mix (Takara); FastStart Universal SYBR Green Master (Rox; Roche, Basel, Switzerland); Luria Bertani broth (Oxoid, Hampshire, UK); Mueller-Hinton II cation adjusted broth (MH broth; BD Biosciences, Franklin Lakes, NJ, USA); and optimal cutting temperature compound (Sakura, Torrance, CA, USA).

Techniques: Infection, Immunofluorescence, Staining, Western Blot, Control