Journal: Mediators of Inflammation

Article Title: DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone

doi: 10.1155/2017/2642549

Figure Lengend Snippet: In the initial two experiments with bovine chondrocytes, HMGB1 synergized with IL-1 β on upregulating metalloproteinase production while MTDs (fMLF and CpG DNA) or HMGB1 alone showed little effect. Cells were harvested from cartilage in bovine stifle joints and cultured for 1 week. Passage 1 cells were treated with MTDs, HMGB1, and/or IL-1 β . After 24 hrs, culture medium was collected, dialyzed, and concentrated. The expression of MMPs in each medium sample was determined with Western blotting. U. C. = untreated control; CpG = CpG-rich DNA; CpG N. C. = CpG-rich DNA negative control.

Article Snippet: This could be attributed to the fact that the recombinant IL-1 β protein used in our study was human origin (R&D Systems, Cat # 201-LB) and might cause less strength of stimulation to bovine cells than to human cells.

Techniques: Cell Culture, Expressing, Western Blot, Control, Negative Control

Journal: Mediators of Inflammation

Article Title: DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone

doi: 10.1155/2017/2642549

Figure Lengend Snippet: Observations made in bovine chondrocytes were further verified in the same type of cells in humans. Articular chondrocytes were isolated from the ankle cartilage of an amputated patient. Passage 2 cells were treated with DAMPs and/or Fn-f (a) or IL-1 β (b) for 24 hrs. Medium samples were analyzed for MMP-3 expression with Western blotting. Relative intensity of protein bands on each blot was measured and plotted, respectively.

Article Snippet: This could be attributed to the fact that the recombinant IL-1 β protein used in our study was human origin (R&D Systems, Cat # 201-LB) and might cause less strength of stimulation to bovine cells than to human cells.

Techniques: Isolation, Expressing, Western Blot

Journal: Mediators of Inflammation

Article Title: DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone

doi: 10.1155/2017/2642549

Figure Lengend Snippet: Observations made in bovine chondrocytes were further verified in the same type of cells in humans. Articular chondrocytes were isolated from ankle cartilage of an amputated patient. Passage 2 cells were treated with DAMPs and/or Fn-f (a) or IL-1 β (b) for 24 hrs. Medium samples were analyzed for expression of MMP-13 and ADAMTS-5 with Western blotting. Furthermore, the induction pattern of ADAM-8, a newly discovered fibronectinase, was examined with the same technique.

Article Snippet: This could be attributed to the fact that the recombinant IL-1 β protein used in our study was human origin (R&D Systems, Cat # 201-LB) and might cause less strength of stimulation to bovine cells than to human cells.

Techniques: Isolation, Expressing, Western Blot

Journal: Mediators of Inflammation

Article Title: DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone

doi: 10.1155/2017/2642549

Figure Lengend Snippet: Protein expression of iNOS, an inflammation-pathway-downstream effector, was only induced by IL-1 β or TNF- α not by DAMPs. Bovine (a) or human (b) articular chondrocytes were treated with DAMPs or cytokines or Fn-f or in combination for 24 hrs. Cells were then lyzed, and the lysates were examined for expression of iNOS with Western blotting.

Article Snippet: This could be attributed to the fact that the recombinant IL-1 β protein used in our study was human origin (R&D Systems, Cat # 201-LB) and might cause less strength of stimulation to bovine cells than to human cells.

Techniques: Expressing, Western Blot

Journal: Mediators of Inflammation

Article Title: DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone

doi: 10.1155/2017/2642549

Figure Lengend Snippet: The synergism between HMGB1 and IL-1 β was replicable in the 3rd experiment with bovine chondrocytes. Such synergism was also observed between DAMPs and TNF- α but to a lesser extent. However, DAMPs were unable to synergize with Fn-f on the upregulation of MMPs while the synergism between cytokines and Fn-f was observed. Moreover, DAMPs alone or in combination did not evoke secretion of MMPs from bovine chondrocytes, which was consistent with what was observed in previous two experiments. Passage 1 bovine chondrocytes were treated with DAMPs with or without Fn-f (a), with or without proinflammatory cytokines (TNF- α or IL-1 β ) (b) for 24 hrs. Culture medium was then examined for MMP expression with Western blotting. In addition, some cells were insulted with culture medium containing soluble substances released from injured cartilage which was bluntly impacted at 7 or 14 J/cm 2 and cultured for 1 day. After 24 hrs of incubation, culture medium was resolved by SDS-PAGE side by side with medium samples from cultures treated with HMGB1, or MTDs, or Fn-f, or TNF- α . Expression of MMP-3 was then examined with immunoblotting (c). U. C. = untreated control; U. I. = unimpacted control; I. = impacted.

Article Snippet: This could be attributed to the fact that the recombinant IL-1 β protein used in our study was human origin (R&D Systems, Cat # 201-LB) and might cause less strength of stimulation to bovine cells than to human cells.

Techniques: Expressing, Western Blot, Cell Culture, Incubation, SDS Page, Control

Journal: Mediators of Inflammation

Article Title: DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone

doi: 10.1155/2017/2642549

Figure Lengend Snippet: Summary of various types of treatment involving DAPMs examined in the study.

Article Snippet: This could be attributed to the fact that the recombinant IL-1 β protein used in our study was human origin (R&D Systems, Cat # 201-LB) and might cause less strength of stimulation to bovine cells than to human cells.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: VSMC isolated from IL-2Rα KO mice express IL-2Rα protein. (A) VSMC, isolated from IL-2Rα WT and KO mice, were cultured to 60 - 70% confluency. After fixation and blocking, primary antibodies recognizing IL-2Rα, purchased from various sources as indicated, were applied to VSMC followed by the appropriate Cy5 conjugated secondary. Cells were imaged using an EVOS FL epifluorescence microscope (ThermoFisher Scientific). Scale bar = 100 μ. (B) Human VSMC were stained with the anti-IL-2Rα from BosterBio. Scale bar = 100 μ. (C) Murine VSMC were stained as in (A) using anti-IL-2Rα from Boster with/without pre-adsorption with immunizing peptide. Scale bar = 200 μ. (D) Lysates of VSMC or Jurkat T cells were separated by SDS-PAGE and analyzed for expression of IL-2Rα, using anti-IL-2Rα from Boster. Histone H3 was used as a loading control. Results shown are representative of >5 (A, B) , 3 (C) , and >5 (D) separate experiments.

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Isolation, Cell Culture, Blocking Assay, Microscopy, Staining, Adsorption, SDS Page, Expressing, Control

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: IL-2Rα is detectable in the nucleus of IL-2Rα WT and KO splenocytes. (A) Splenocytes, prepared fresh (untreated/no PHA) or following 48h of stimulation with PHA 10 μg/ml, were stained with anti-IL-2Rα antibodies or isotype controls as indicated with/without first permeabilizing via methanol. Graphs to the right of histograms represent a summary of individual values from multiple experiments. Permeabilized (perm) groups with/without PHA were combined. (B) represents the average MFIs ± SEM, derived from (A) , of permeabilized splenocytes stained with anti-IL-2Rα antibodies as indicated. (C) Splenocytes were stimulated with PHA for 48h, pelleted and fractionated into membrane, cytoplasm, and nuclear fractions, then analyzed by Western blot probing with antibodies as indicated. Blots represent lysates processed in parallel. Total protein, obtained from stain-free gels (Bio-Rad), shows the single most prominent band present across all samples. Results shown are representative of ≥ 3 experiments. * P ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Staining, Derivative Assay, Membrane, Western Blot

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: IL-2Rα is detected in permeabilized splenocytes by rat anti-mouse IL-2Rα (clone PC 61) following DNA digestion. (A) Splenocytes were prepared as described in Figure 2 . A subset of methanol permeabilized cells were treated with Benzonase ® 250U/10 6 cells or enzyme buffer for 50 minutes at 37°C prior to staining. In a subset of samples, anti-IL-2Rα/clone PC 61 was pre-adsorbed with an excess of purified mouse IL-2Rα. Graphs to the right of histograms represent a summary of individual values from multiple experiments. “Block” indicates use of pre-adsorbed anti-IL-2Rα. (B) represents the average MFIs ± SEM, derived from (A) , of permeabilized splenocytes treated with Benzonase ® . (C) VSMC were permeabilized with methanol and treated with Benzonase ® or buffer, as indicated above, then stained with anti-IL-2Rα/clone PC 61. Note that digestion with Benzonase ® eliminates DAPI staining as expected. Scale bar = 100 μ. *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Staining, Purification, Blocking Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: Exons 2 and 3 of IL-2Rα gene are deleted in IL-2Rα KO mice. Genomic DNA isolated from WT and KO VSMC underwent targeted sequencing, with a focus on the IL-2Rα locus. Exons 2 and 3 of IL-2Rα are deleted as reported by Willerford, et al. .

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Isolation, Sequencing

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: IL-2Rα gene is present in IL-2Rα KO tissues. Genomic DNA was isolated from various organs as indicated. Levels of IL2RA (A) or Neo resistance (B) DNA were measured in each tissue by qPCR. The relative quantity (RQ) of IL2RA or Neo DNA was measured relative to a single KO spleen (A) or a WT spleen (B) .

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Isolation

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: G418 decreases IL-2Rα protein detected in VSMC isolated from IL-2Rα KO mice. (A) VSMC, isolated from IL-2Rα WT and KO mice, were cultured in the presence/absence of G418 at 2 mg/ml. Cells were probed for IL-2Rα expression using the anti-IL-2Rα from Boster as described in Figure 1 . Scale bar = 400 μ. (B) The intensity of IL-2Rα staining in all cells from (A) was measured in an image cytometer and converted to a histogram using flow cytometry software. To highlight differences in fluorescence intensity, histograms were normalized to peak values of WT or KO cells at 0 mg/ml G418. Cell numbers analyzed, in order from 0 – 2 mg/ml G418, were as follows: WT 7425, 911, 218; KO 7621, 9941, 7656. (C) KO VSMC, cultured as described in (A) , were lysed. Extracted proteins were separated by SDS-PAGE and analyzed by Western blot for expression of IL-2Rα using the anti-IL-2Rα antibody from Boster. Intensity of the IL-2Rα band from each treatment was expressed as a ratio of IL-2Rα/histone H3. Densitometry was performed using Image Lab software from Bio-Rad Laboratories. Results shown for (A, C) are representative of >3,2 separate experiments respectively.

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Isolation, Cell Culture, Expressing, Staining, Cytometry, Flow Cytometry, Software, Fluorescence, SDS Page, Western Blot

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: IL-2Rα is transferred between cells. (A) VSMC were cultured for 96h with increasing concentrations of G418 as indicated. IL-2Rα protein expression was detected using the anti-IL-2Rα antibody from BosterBio. (B) VSMC, cultured in G418, were separated by size using 1 and 30 micron filters as indicated. Genomic DNA was isolated from cell pellets and levels of IL2RA DNA were quantified relative to the 1 micron cells. (C) Human VSMC were placed in transwell inserts and co-cultured with murine VSMC for 96h and compared to murine VSMC without co-culture. IL-2Rα protein expression was detected using the anti-IL-2Rα antibody from BosterBio. Intensity of IL-2Rα staining in all cells was measured in an image cytometer (Cytation) then converted to a histogram or bar graph using flow cytometry or data analysis software. Scale bar = 200 μ. **** P ≤ 0.0001.

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Cell Culture, Expressing, Isolation, Co-Culture Assay, Staining, Cytometry, Flow Cytometry, Software

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: IL-2Rα exhibits a prolonged half-life. VSMC were labeled with AHA-containing, methionine free media for 48h then chased with complete media for the durations above. IL-2Rα was then isolated by immunoprecipitation with anti-IL-2Rα from BosterBio. AHA-labeled IL-2Rα was detected by DBCO-488 and total IL-2Rα was detected using anti-IL-2Rα, anti-phospho IL-2Rα ser268, or total protein stain as indicated. DBCO intensities, normalized to total IL-2Rα, were graphed on a linear scale and linear regression analysis was performed to determine the protein half-life. Blots at the left and right edges of the figure show VSMC fractionated into membrane, soluble nuclear, and insoluble nuclear fractions (both nuclear fractions digested with Benzonase ® ) and probed with antibodies as indicated. Circled lanes indicate the fractions that were combined for immunoprecipitation. The half-life on the left hand side of the figure was calculated using the band detected by anti-phospho IL-2Rα ser268 and total protein (of that band) for normalization. The half-life on the right hand side was calculated using the band detected by anti-IL-2Rα from BosterBio and immunodetection for normalization. In the above experiments, immunoprecipitated IL-2Rα was eluted using a denaturing buffer. Use of a non-denaturing buffer yielded a similar half-life of 8.25 days.

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Labeling, Isolation, Immunoprecipitation, Staining, Membrane, Immunodetection

Journal: Frontiers in Immunology

Article Title: IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

doi: 10.3389/fimmu.2024.1369818

Figure Lengend Snippet: IL-2Rα KO VSMC are small and hypodiploid. (A) VSMC, isolated from IL-2Rα WT and KO mice, were cultured for 72h in serum free media with increasing concentrations of FBS. Cells were probed for IL-2Rα expression using the anti-IL-2Rα from Boster as described in Figure 1 . Scale bar = 200 μ. (B) Doubling times of WT and KO VSMC were calculated using three time points over 72h . Data represent average ± SEM of 5 separate experiments. ( C , top) Phase and IL-2Rα overlay images from WT and KO VSMC in serum free media and probed for IL-2Rα as in (A) . Scale bar = 100 μ. ( C , middle and bottom) Nuclei of VSMC from (A) were imaged for area and DAPI intensity using a Cytation imaging plate reader (Biotek). Data accrued from these images were then processed using flow cytometry software. Results shown are representative of 3 (A, C) separate experiments or 5 (B) collated experiments. ** P ≤ 0.01.

Article Snippet: Levels of DBCO-labeled IL-2Rα were normalized using either total protein (RevertTM 700 Total Protein Stain) or total IL-2Rα detected by either the anti-IL-2Rα antibody from BosterBio or anti-phospho-CD25 (ser268).

Techniques: Isolation, Cell Culture, Expressing, Imaging, Flow Cytometry, Software