Journal: Animal Nutrition

Article Title: Different copper sources and levels affect growth performance, copper content, carcass characteristics, intestinal microorganism and metabolism of finishing pigs

doi: 10.1016/j.aninu.2021.10.007

Figure Lengend Snippet: Effects of different copper sources and levels on the mRNA expression of CTR1 (A), ATP7A (B), ATOX1 (C), ASCT2 (D) and PepT1 (E) genes in jejunal mucosa. CuSO 4 = copper sulfate; Cu-Gly = copper glycinate; Cu-Pro = copper proteinate; CTR1 = high affinity copper uptake protein 1; ATP7A = ATPase copper transporting alpha; ATOX1 = Antioxidant 1 copper chaperone; ASCT2 = lanine-serine-cysteine transporter, type-2; PepT1 = peptide transporter 1. Data represent mean values ± standard error of the mean ( n = 6). Significant differences between processing treatments are represented by different lowercase letters ( P < 0.05).

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) and incubated overnight at 4 °C with the antibodies: rabbit anti-CTR1 (1:1,000, Genetex, GTX30642), rabbit anti-PepT1 (1:1,000, Bioss, bs-0689R) and rabbit anti-actin (1:1,000, Absin, abs132001).

Techniques: Expressing

Journal: Animal Nutrition

Article Title: Different copper sources and levels affect growth performance, copper content, carcass characteristics, intestinal microorganism and metabolism of finishing pigs

doi: 10.1016/j.aninu.2021.10.007

Figure Lengend Snippet: Effects of different copper sources and levels on the mRNA Expression of CTR1 (A), ATP7A (B), ATP7B (C), ATOX1 (D), ASCT2 (E) and PepT1 (F) genes in liver. CuSO 4 = copper sulfate; Cu-Gly = copper glycinate; Cu-Pro = copper proteinate; CTR1 = high affinity copper uptake protein 1; ATP7A = ATPase copper transporting alpha; ATOX1 = Antioxidant 1 copper chaperone; ASCT2 = lanine-serine-cysteine transporter, type-2; PepT1 = peptide transporter 1. Data represent mean values ± standard error of the mean ( n = 6). Significant differences between processing treatments are represented by different lowercase letters ( P < 0.05).

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) and incubated overnight at 4 °C with the antibodies: rabbit anti-CTR1 (1:1,000, Genetex, GTX30642), rabbit anti-PepT1 (1:1,000, Bioss, bs-0689R) and rabbit anti-actin (1:1,000, Absin, abs132001).

Techniques: Expressing

Journal: Animal Nutrition

Article Title: Different copper sources and levels affect growth performance, copper content, carcass characteristics, intestinal microorganism and metabolism of finishing pigs

doi: 10.1016/j.aninu.2021.10.007

Figure Lengend Snippet: Effects of different copper sources and levels on the protein expression for CTR1(A), and PepT1(B) in jejunal mucosa. CuSO 4 = copper sulfate; Cu-Gly = copper glycinate; Cu-Pro = copper proteinate; CTR1 = high affinity copper uptake protein 1; PepT1 = peptide transporter 1. Data represent mean values ± standard error of the mean ( n = 6). Significant differences between processing treatments are represented by different lowercase letters ( P < 0.05).

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) and incubated overnight at 4 °C with the antibodies: rabbit anti-CTR1 (1:1,000, Genetex, GTX30642), rabbit anti-PepT1 (1:1,000, Bioss, bs-0689R) and rabbit anti-actin (1:1,000, Absin, abs132001).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Regulation of CYP450 and drug transporter mediated by gut microbiota under high-altitude hypoxia

doi: 10.3389/fphar.2022.977370

Figure Lengend Snippet: Primer sequences corresponding to the rat genes examined with a RT-qPCR analysis.

Article Snippet: Anti-mouse β-actin (1:500 dilution, Abcam, lot: ab8226), anti-mouse CYP1A2 (1:1000 dilution, Abcam, lot: ab22717), anti-mouse CYP2B1 (1:1000 dilution, Thermo Fisher, lot: MA5-25882), anti-rabbit CYP2C11 (1:1000 dilution, Biorbyt, lot: orb5951), anti-rabbit CYP2E1 (1:1000 dilution, Abcam, lot: ab28146), anti-rabbit CYP3A1 (1:1000 dilution, Abcam, lot: ab3572), anti-rabbit BCRP (1:500 dilution, Abcam, lot: ab207732), anti-rabbit MDR1 (1:1000 dilution, Abcam, lot: ab170904), anti-rabbit MRP2 (1:1000 dilution, Sigma-Aldrich, lot: M8316), anti-rabbit OATP2B1 (1:500 dilution, Novus, lot: NBP-1-59811), anti-rabbit OCT1 (1:1000 dilution, Abcam, lot: ab178869), and anti-rabbit PEPT1 (1:500 dilution, Thermo Fisher, lot: PA5-37010) antibodies were used for Western blotting.

Techniques: Sequencing

Journal: Frontiers in Physiology

Article Title: Organic zinc with moderate chelation strength enhances zinc absorption in the small intestine and expression of related transporters in the duodenum of broilers

doi: 10.3389/fphys.2022.952941

Figure Lengend Snippet: Primer sequences for real-time PCR amplification.

Article Snippet: Western blotting analyses for rabbit anti-human ZnT1 (ab214356, Abcam, diluted 1:1,000), rabbit anti-mouse ZnT4 (BA3484, Boster, diluted 1:1,000), rabbit anti-human ZnT5 (25604-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT7 (13966-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT9 (BS62531, Bioworld, diluted 1:1,000), rabbit anti-human ZnT10 (ab229954, Abcam, diluted 1:1,000), rabbit anti-human ZIP3 (ab254868, Abcam, diluted 1:500), rabbit anti-human ZIP5 (ab105194, Abcam, diluted 1:1,000), rabbit anti-human B 0 AT1 (ab180516, Abcam, diluted 1:1,000), rabbit anti-human LAT1 (DF8065, Affinity Biosciences, diluted 1:500), rabbit anti-human y + LAT2 (13823-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human rBAT (DF7379, Affinity Biosciences, diluted 1:1,000) and rabbit anti-human PepT1 (A03672-1, Boster, diluted 1:500) were carried out according to the recommended protocols provided by the manufacturers.

Techniques: Real-time Polymerase Chain Reaction, Amplification, Sequencing

Journal: Frontiers in Physiology

Article Title: Organic zinc with moderate chelation strength enhances zinc absorption in the small intestine and expression of related transporters in the duodenum of broilers

doi: 10.3389/fphys.2022.952941

Figure Lengend Snippet: Effect of dietary Zn source on the mRNA expression levels of Zn, amino acid, and small peptide transporters in the duodenum of broilers at 28 days of age. Values are means ± SE, ( n = 7/8). Lacking the same letters (a, b, and c) means significant differences, p < 0.05. ZnT1, zinc transporter 1; ZnT4, zinc transporter 4; ZnT5, zinc transporter 5; ZnT7, zinc transporter 7; ZnT9, zinc transporter 9; ZIP3, Zrt-irt-like protein 3; ZIP5, Zrt-irt-like protein 5; B0AT1, B-0-system neutral amino acid co-transporter; LAT1, L-type amino acid transporter 1; y + LAT2, y + L-type amino acid transporter 2; rBAT, b0,+-type amino acid transporter; EAAT3, excitatory amino acid transporter 3; PepT1, peptide-transporter 1; Zn-Prot M, Zn proteinate with moderate chelation strength (Q f = 51.6). The mRNA expression levels were calculated as the relative quantities (RQs) of the target gene mRNA to the geometric mean of β-actin and GAPDH mRNA using the 2 −ΔΔCT method.

Article Snippet: Western blotting analyses for rabbit anti-human ZnT1 (ab214356, Abcam, diluted 1:1,000), rabbit anti-mouse ZnT4 (BA3484, Boster, diluted 1:1,000), rabbit anti-human ZnT5 (25604-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT7 (13966-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT9 (BS62531, Bioworld, diluted 1:1,000), rabbit anti-human ZnT10 (ab229954, Abcam, diluted 1:1,000), rabbit anti-human ZIP3 (ab254868, Abcam, diluted 1:500), rabbit anti-human ZIP5 (ab105194, Abcam, diluted 1:1,000), rabbit anti-human B 0 AT1 (ab180516, Abcam, diluted 1:1,000), rabbit anti-human LAT1 (DF8065, Affinity Biosciences, diluted 1:500), rabbit anti-human y + LAT2 (13823-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human rBAT (DF7379, Affinity Biosciences, diluted 1:1,000) and rabbit anti-human PepT1 (A03672-1, Boster, diluted 1:500) were carried out according to the recommended protocols provided by the manufacturers.

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Organic zinc with moderate chelation strength enhances zinc absorption in the small intestine and expression of related transporters in the duodenum of broilers

doi: 10.3389/fphys.2022.952941

Figure Lengend Snippet: Effect of dietary Zn source on the mRNA expression levels of Zn, amino acid, and small peptide transporters in the duodenum of broilers at 39 days of age. Values are means ± SE, (n = 7/8). Lacking the same letters (a and b) means significant differences, p < 0.05. ZnT1, zinc transporter 1; ZnT4, zinc transporter 4; ZnT5, zinc transporter 5; ZnT7, zinc transporter 7; ZnT9, zinc transporter 9; ZIP3, Zrt-irt-like protein 3; ZIP5, Zrt-irt-like protein 5; B0AT1, B-0-system neutral amino acid co-transporter; LAT1, L-type amino acid transporter 1; y + LAT2, y + L-type amino acid transporter 2; rBAT, b0,+-type amino acid transporter; EAAT3, excitatory amino acid transporter 3; PepT1, peptide-transporter 1; Zn-Prot M, Zn proteinate with moderate chelation strength (Q f = 51.6). The mRNA expression levels were calculated as the relative quantities (RQs) of the target gene mRNA to the geometric mean of β-actin and GAPDH mRNA using the 2 −ΔΔCT method.

Article Snippet: Western blotting analyses for rabbit anti-human ZnT1 (ab214356, Abcam, diluted 1:1,000), rabbit anti-mouse ZnT4 (BA3484, Boster, diluted 1:1,000), rabbit anti-human ZnT5 (25604-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT7 (13966-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT9 (BS62531, Bioworld, diluted 1:1,000), rabbit anti-human ZnT10 (ab229954, Abcam, diluted 1:1,000), rabbit anti-human ZIP3 (ab254868, Abcam, diluted 1:500), rabbit anti-human ZIP5 (ab105194, Abcam, diluted 1:1,000), rabbit anti-human B 0 AT1 (ab180516, Abcam, diluted 1:1,000), rabbit anti-human LAT1 (DF8065, Affinity Biosciences, diluted 1:500), rabbit anti-human y + LAT2 (13823-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human rBAT (DF7379, Affinity Biosciences, diluted 1:1,000) and rabbit anti-human PepT1 (A03672-1, Boster, diluted 1:500) were carried out according to the recommended protocols provided by the manufacturers.

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Organic zinc with moderate chelation strength enhances zinc absorption in the small intestine and expression of related transporters in the duodenum of broilers

doi: 10.3389/fphys.2022.952941

Figure Lengend Snippet: Effect of dietary Zn source on the protein expression levels of Zn, amino acid, and small peptide transporters in the duodenum of broilers at 28 days of age. Values are means ± SE, (n = 7/8). Lacking the same letters (a, b, and c) means significant differences, p < 0.05. ZnT1, zinc transporter 1; ZnT4, zinc transporter 4; ZnT5, zinc transporter 5; ZnT7, zinc transporter 7; ZnT9, zinc transporter 9; ZIP3, Zrt-irt-like protein 3; ZIP5, Zrt-irt-like protein 5; B0AT1, B-0-system neutral amino acid co-transporter; LAT1, L-type amino acid transporter 1; y + LAT2, y + L-type amino acid transporter 2; rBAT, b0,+-type amino acid transporter; PepT1, peptide-transporter 1; Zn-Prot M, Zn proteinate with moderate chelation strength (Q f = 51.6). The protein expression levels were calculated as the relative quantities (RQs) of the target gene protein to the β-tubulin protein.

Article Snippet: Western blotting analyses for rabbit anti-human ZnT1 (ab214356, Abcam, diluted 1:1,000), rabbit anti-mouse ZnT4 (BA3484, Boster, diluted 1:1,000), rabbit anti-human ZnT5 (25604-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT7 (13966-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT9 (BS62531, Bioworld, diluted 1:1,000), rabbit anti-human ZnT10 (ab229954, Abcam, diluted 1:1,000), rabbit anti-human ZIP3 (ab254868, Abcam, diluted 1:500), rabbit anti-human ZIP5 (ab105194, Abcam, diluted 1:1,000), rabbit anti-human B 0 AT1 (ab180516, Abcam, diluted 1:1,000), rabbit anti-human LAT1 (DF8065, Affinity Biosciences, diluted 1:500), rabbit anti-human y + LAT2 (13823-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human rBAT (DF7379, Affinity Biosciences, diluted 1:1,000) and rabbit anti-human PepT1 (A03672-1, Boster, diluted 1:500) were carried out according to the recommended protocols provided by the manufacturers.

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Organic zinc with moderate chelation strength enhances zinc absorption in the small intestine and expression of related transporters in the duodenum of broilers

doi: 10.3389/fphys.2022.952941

Figure Lengend Snippet: Effect of dietary Zn source on the protein expression levels of Zn, amino acid, and small peptide transporters in the duodenum of broilers at 39 days of age. Values are means ± SE, (n = 7/8). Lacking the same letters (a and b) means significant differences, p < 0.05. ZnT1, zinc transporter 1; ZnT4, zinc transporter 4; ZnT5, zinc transporter 5; ZnT7, zinc transporter 7; ZnT9, zinc transporter 9; ZIP3, Zrt-irt-like protein 3; ZIP5, Zrt-irt-like protein 5; B0AT1, B-0-system neutral amino acid co-transporter; LAT1, L-type amino acid transporter 1; y + LAT2, y + L-type amino acid transporter 2; rBAT, b0,+-type amino acid transporter; PepT1, peptide-transporter 1; Zn-Prot M, Zn proteinate with moderate chelation strength (Qf = 51.6). The protein expression levels were calculated as the relative quantities (RQs) of the target gene protein to the β-tubulin protein.

Article Snippet: Western blotting analyses for rabbit anti-human ZnT1 (ab214356, Abcam, diluted 1:1,000), rabbit anti-mouse ZnT4 (BA3484, Boster, diluted 1:1,000), rabbit anti-human ZnT5 (25604-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT7 (13966-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human ZnT9 (BS62531, Bioworld, diluted 1:1,000), rabbit anti-human ZnT10 (ab229954, Abcam, diluted 1:1,000), rabbit anti-human ZIP3 (ab254868, Abcam, diluted 1:500), rabbit anti-human ZIP5 (ab105194, Abcam, diluted 1:1,000), rabbit anti-human B 0 AT1 (ab180516, Abcam, diluted 1:1,000), rabbit anti-human LAT1 (DF8065, Affinity Biosciences, diluted 1:500), rabbit anti-human y + LAT2 (13823-1-AP, Proteintech, diluted 1:1,000), rabbit anti-human rBAT (DF7379, Affinity Biosciences, diluted 1:1,000) and rabbit anti-human PepT1 (A03672-1, Boster, diluted 1:500) were carried out according to the recommended protocols provided by the manufacturers.

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: POT transporter expression in mouse bone marrow. Gene expression of Pept1 (A), protein expression of PEPT1 (B), gene expression of Pept2 (C), gene expression of Pht1 (D), gene expression of Pht2 (E), and protein expression of PEPT2 (F) in wildtype, Pht1 knockout (KO) and Pept2 knockout (KO) mice. The positive control was small intestine for PEPT1 and kidney for PEPT2. Quantification of protein (i.e., transporter/β-actin ratio) is shown below the figure. One representative example is shown of three individual immunoblots.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Knock-Out, Positive Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Protein expression of PEPT2 in cell subtypes of mouse bone marrow. PEPT2 protein was examined in (A) Lin− (includes hematopoietic and mesenchymal stem cells, and their progenitors) and Lin+ (includes T and B cells, macrophages, monocytes, granulocytes, erythrocytes and their committed precursors) preparations, and (B) residential macrophages and bone marrow-derived macrophages (BMDMs) isolated from wildtype mice. The positive control was wildtype mouse kidney for panel A and bone marrow for panel B (which was validated in Fig. 1F). Quantification of protein (i.e., transporter/β-actin ratio) is shown below each figure. One representative example is shown of three individual immunoblots.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Derivative Assay, Isolation, Positive Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: GlySar and MDP-rhodamine uptake studies in BMDMs of wildtype and Pept2 knockout mouse. (A) effect of potential inhibitors on the uptake of 1.0 μM [3H]GlySar where GlyPro or glycine were present at 5 mM, and MDP (L,D-isomer), MDP control (L,L-isomer), r-iE-DAP or tri-DAP were present at 1 mM. GlySar uptakes were corrected for nonspecific binding using [14C]mannitol, and represented as % control, relative to wildtype mice. Data are expressed as mean ± SE (n=6) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (B) uptake of MDP-rhodamine in wildtype and Pept2 knockout mice. MDP-rhodamine (red) was marked by arrows and the nuclei (blue) were stained by DAPI (40× magnification). Density measurements are shown in the right-hand panel (LabWorks Image Acquisition and Analysis Software v4.5, UVP Inc, Upland, CA), and expressed as mean ± SE (n=10 cells). Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Knock-Out, Binding Assay, Staining, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Effect of PEPT2 on the expression of IL-6 and TNF-α in BMDMs of wildtype and Pept2 knockout mice. BMDMs were treated for 24 hr with 0–10 ng/mL LPS plus 10 μg/mL MDP (L,D-isomer; A and B), tri-DAP (C and D), or 10 ng/mL LPS plus 10 μg/mL MDP control (L,L-isomer; E and F), after which cytokine concentrations in the cell culture media were measured. Data are expressed as mean ± SE (n=4) in which each experiment was performed in triplicate. Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Knock-Out, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Effect of PHT1 on the LPS-MDP stimulated immune response, and uptake studies of MDP or L-histidine. (A) BMDMs from wildtype and Pht1 knockout mice were treated for 24 hr with 5 ng/mL LPS plus 10 μg/mL MDP, after which IL-6 and TNF-α concentrations in the cell culture media were measured. Data are expressed as mean ± SE (n=6) in which each experiment was performed in triplicate. Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001. (B) BMDMs from wildtype mice were treated for 24 hr with 5 ng/mL LPS alone, and in the presence of 1.0 μM bafilomycin A1 (BAF), 10 μg/mL MDP, or 10 μg/mL MDP plus 1 μM BAF. BMDMs were pretreated with BAF for 30 min prior to being co-incubated with MDP and/or LPS. IL-6 concentrations in the cell culture media were then measured. Data are expressed as mean ± SE (n=3) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (C) uptake of MDP-rhodamine in BMDMs prepared from wildtype and Pht1 knockout mice. MDP-rhodamine (red) was marked by arrows and the nuclei (blue) were stained by DAPI (100× magnification). Particle density is shown in the right-hand panel and expressed as mean ± SE (n=6–8 cells). Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001. (D) effect of potential inhibitors on the uptake of 1.0 μM [3H]histidine into endosome-enriched liver preparations from wildtype and Pht1 knockout mice. Endosomes were pretreated with MDP (1.0 mM) or BAF (1.0 μM) for 30 min prior to experimentation. Data are expressed as mean ± SE (n=3) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (E) fusion protein of GFP-mPHT1 (90 kd) was detected with an anti-GFP monoclonal antibody in the endosome of pcDNA3.1-mPht1/CT-GFP transfected HEK293 cells, but not in pcDNA3.1-CT-GFP transfected HEK293 (mock) cells. (F) expression of PEPT2 protein in mouse liver endosomes. One representative example is shown of three individual immunoblots.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Knock-Out, Cell Culture, Incubation, Staining, Transfection, Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Schematic depicting the PEPT2- and PHT1-mediated uptake of bacterially-derived products (e.g., MDP and tri-DAP) into the cytosol of macrophages, and their effect on NOD signaling and downstream production of proinflammatory cytokines.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Derivative Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: POT transporter expression in mouse bone marrow. Gene expression of Pept1 (A), protein expression of PEPT1 (B), gene expression of Pept2 (C), gene expression of Pht1 (D), gene expression of Pht2 (E), and protein expression of PEPT2 (F) in wildtype, Pht1 knockout (KO) and Pept2 knockout (KO) mice. The positive control was small intestine for PEPT1 and kidney for PEPT2. Quantification of protein (i.e., transporter/β-actin ratio) is shown below the figure. One representative example is shown of three individual immunoblots.

Article Snippet: The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Gene Expression, Knock-Out, Positive Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Protein expression of PEPT2 in cell subtypes of mouse bone marrow. PEPT2 protein was examined in (A) Lin− (includes hematopoietic and mesenchymal stem cells, and their progenitors) and Lin+ (includes T and B cells, macrophages, monocytes, granulocytes, erythrocytes and their committed precursors) preparations, and (B) residential macrophages and bone marrow-derived macrophages (BMDMs) isolated from wildtype mice. The positive control was wildtype mouse kidney for panel A and bone marrow for panel B (which was validated in Fig. 1F). Quantification of protein (i.e., transporter/β-actin ratio) is shown below each figure. One representative example is shown of three individual immunoblots.

Article Snippet: The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Derivative Assay, Isolation, Positive Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: GlySar and MDP-rhodamine uptake studies in BMDMs of wildtype and Pept2 knockout mouse. (A) effect of potential inhibitors on the uptake of 1.0 μM [3H]GlySar where GlyPro or glycine were present at 5 mM, and MDP (L,D-isomer), MDP control (L,L-isomer), r-iE-DAP or tri-DAP were present at 1 mM. GlySar uptakes were corrected for nonspecific binding using [14C]mannitol, and represented as % control, relative to wildtype mice. Data are expressed as mean ± SE (n=6) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (B) uptake of MDP-rhodamine in wildtype and Pept2 knockout mice. MDP-rhodamine (red) was marked by arrows and the nuclei (blue) were stained by DAPI (40× magnification). Density measurements are shown in the right-hand panel (LabWorks Image Acquisition and Analysis Software v4.5, UVP Inc, Upland, CA), and expressed as mean ± SE (n=10 cells). Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001.

Article Snippet: The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Knock-Out, Control, Binding Assay, Staining, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Effect of PEPT2 on the expression of IL-6 and TNF-α in BMDMs of wildtype and Pept2 knockout mice. BMDMs were treated for 24 hr with 0–10 ng/mL LPS plus 10 μg/mL MDP (L,D-isomer; A and B), tri-DAP (C and D), or 10 ng/mL LPS plus 10 μg/mL MDP control (L,L-isomer; E and F), after which cytokine concentrations in the cell culture media were measured. Data are expressed as mean ± SE (n=4) in which each experiment was performed in triplicate. Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

Article Snippet: The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Knock-Out, Control, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Effect of PHT1 on the LPS-MDP stimulated immune response, and uptake studies of MDP or L-histidine. (A) BMDMs from wildtype and Pht1 knockout mice were treated for 24 hr with 5 ng/mL LPS plus 10 μg/mL MDP, after which IL-6 and TNF-α concentrations in the cell culture media were measured. Data are expressed as mean ± SE (n=6) in which each experiment was performed in triplicate. Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001. (B) BMDMs from wildtype mice were treated for 24 hr with 5 ng/mL LPS alone, and in the presence of 1.0 μM bafilomycin A1 (BAF), 10 μg/mL MDP, or 10 μg/mL MDP plus 1 μM BAF. BMDMs were pretreated with BAF for 30 min prior to being co-incubated with MDP and/or LPS. IL-6 concentrations in the cell culture media were then measured. Data are expressed as mean ± SE (n=3) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (C) uptake of MDP-rhodamine in BMDMs prepared from wildtype and Pht1 knockout mice. MDP-rhodamine (red) was marked by arrows and the nuclei (blue) were stained by DAPI (100× magnification). Particle density is shown in the right-hand panel and expressed as mean ± SE (n=6–8 cells). Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001. (D) effect of potential inhibitors on the uptake of 1.0 μM [3H]histidine into endosome-enriched liver preparations from wildtype and Pht1 knockout mice. Endosomes were pretreated with MDP (1.0 mM) or BAF (1.0 μM) for 30 min prior to experimentation. Data are expressed as mean ± SE (n=3) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (E) fusion protein of GFP-mPHT1 (90 kd) was detected with an anti-GFP monoclonal antibody in the endosome of pcDNA3.1-mPht1/CT-GFP transfected HEK293 cells, but not in pcDNA3.1-CT-GFP transfected HEK293 (mock) cells. (F) expression of PEPT2 protein in mouse liver endosomes. One representative example is shown of three individual immunoblots.

Article Snippet: The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Knock-Out, Cell Culture, Incubation, Staining, Transfection, Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Schematic depicting the PEPT2- and PHT1-mediated uptake of bacterially-derived products (e.g., MDP and tri-DAP) into the cytosol of macrophages, and their effect on NOD signaling and downstream production of proinflammatory cytokines.

Article Snippet: The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Derivative Assay