Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 5. NaF exposure induces elevated phos phorylation of PRKAA1 expression in vivo and in vitro. (A) The hierarchical cluster heatmap of phosphorylated proteins in rat hippocampus. (B) Statistics of different phosphorylation sites and proteins in rat hippocampus. (C) Volcano plot of differentially phosphorylated proteins in rat hippocampus. The criteria for significance were set at logarithmic fold change (log2 FC) of > 1.2 or < 0.8 (P < 0.05). (D) Phosphorylation of PRKAA1 protein in rat hippocampus. (E-F) Representative western blot and relative quan tifications of PRKAA1 and p-PRKAA1 in rat hippocampus. (G-H) Representative western blot and relative quantifications of PRKAA1 and p-PRKAA1 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Expressing, In Vivo, In Vitro, Phospho-proteomics, Western Blot, Control

Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 6. DM inhibits aberrant mitophagy by pre venting the phosphorylation of PRKAA1 in vitro. (A-B) Representative western blot and relative quantifications of PRKAA1and p-PRKAA1 in SH- SY5Y cells. (C-D) Representative western blot and relative quantifications of PINK1 and Parkin in SH-SY5Y cells. (E-F) Representative images and relative quantifications of immunofluores cence staining of PINK1 and Mito-tracker, after DM intervention. The scale bar represents 50 µm. (G-H) Representative western blot and relative quantifications of TOMM-20 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group, @P < 0.05 compare with the NaF group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Phospho-proteomics, In Vitro, Western Blot, Staining, Control

Journal: Cancer Medicine

Article Title: The expression and role of SUZ12 in lung adenocarcinoma

doi: 10.1002/cam4.70190

Figure Lengend Snippet: The effect of SUZ12 on metastasis related genes expression was tested by qRT‐PCR and western blotting. sh‐SUZ12 increased MMP1/2/3/9 mRNA expression (A) and TIMP1/2 protein expression (B), while decreased MMP14 mRNA expression (A), MMP1/9/14 (B), TIMP3 (B), and ITGB1/5(F) protein expression, without significantly effected ITGBs mRNA expression (E). oe‐SUZ12 increased MMP14 mRNA (C) and protein (D) expression, while decreased TIMP2 mRNA expression (C) and TIMP1/2 (D) protein expression.

Article Snippet: The list of primary antibodies: SUZ12 (1 μg/mL, Abcam Cambridge, cat no: ab12073), CDK2 (1:1000; Proteintech, USA, cat. no. 10122‐1‐AP), CDK3 (1:2000; Proteintech, USA, cat. no. 55103‐1‐AP), CDK6 (1:2000; Proteintech, USA, cat. no. 14052‐1‐AP), cyclin D1 (1:5000; Proteintech, USA, cat. no. 26939‐1‐AP), cyclin E1 (1:1000; Proteintech, USA, cat. no. 11554‐1‐AP), p18 (1:1000, BOSTER China, cat. no. M03299‐1), p19 (1:1000, BOSTER China, cat. no. MA1075), p53 (1:5000; Proteintech, USA, cat. no. 60283‐2‐Ig), p‐p53 (1:2000; Proteintech, USA, cat. no. 28961‐1‐AP), p57 (1:1000, BOSTER China, cat. no. BM4129), Rb (1:1000, BOSTER China, cat. no. BM4500), pRb (1:1000, BOSTER China, cat. no. BM4338), Bcl‐2 (1:1000; Proteintech, USA, cat. no. 26593‐1‐AP), Bax (1:2000; Proteintech, USA, cat. no. 50599‐2‐lg), E‐cadherin (1:5000; Proteintech, USA, cat. no. 20874‐1‐AP), N‐cadherin (1:3000; Proteintech, USA, cat. no. 22018‐1‐AP), vimentin (1:4000; Proteintech, USA, cat. no. 10366‐1‐AP), MMP1 (1:1000, BOSTER China, cat. no. A00733‐1), MMP2 (1:500, BOSTER China, cat. no. BM4075), MMP9 (1:1000, BOSTER China, cat. no. PB0709), MMP14 (1:1000, BOSTER China, cat. no. BM4119), TIMP1 (1:1000, Bioss China, cat. no. bs‐0415R), TIMP2 (1:1000, Bioss China, cat. no. bs‐10395R), TIMP3 (1:1000; Proteintech, USA, cat. no. 10858‐1‐AP), ITGB1 (1:1000, BOSTER China, cat. no. BM4308), ITGB3 (1:1000, BOSTER China, cat. no. BA1670), ITGB5 (1:1000, BOSTER China, cat. no. A04201‐1), nm23 (1:1000, BOSTER China, cat. no. BA3787), PD‐L1 (1:3000; Proteintech, USA, cat. no. 66248‐1‐Ig), and β‐actin (1:5000; Proteintech, USA, cat. no. 66009‐1‐Ig).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: DRI-Pep #20 is a potent PI3Kγ/PKA disruptor peptide. A , chemical structure of DRI-Pep #20. The amino acid sequence of DRI-Pep #20 comprises the nonnatural D-peptide RHQGK, the D-retroinverso (DRI)-isoform of the cell penetrating peptide Penetratin 1 (P1) and a glycine (G) linker. B , schematic representation of the fluorescence spectroscopy assays for the characterization of the interaction between DRI-Pep #20 (or PI3Kγ MP) and the recombinant fluorescein 5-maleimide–labeled PKA-RIIα (PKA-F5M). C , steady-state emission spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep #20 (0–20 μM). K D : dissociation constant. Inset, nonlinear fitting of the fluorescence intensity maxima obtained at various concentrations of DRI-Pep #20 for the monitoring of bio-labeled PKA. K A : association constant. D , for kinetic analysis, fluorescence spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep #20 or PI3Kγ MP (inset) were analyzed and fitted to a single exponential function to obtain the observed rate constant ( k obs ). The binding of DRI-Pep #20 or PI3Kγ MP to biolabeled PKA was investigated under pseudo -first-order conditions, and the kinetic constants, k on and k off , were determined. E , schematic representation of the displacement assay between DRI-Pep #20 (or PI3Kγ MP) and the PI3Kγ/PKA-F5M complex. F , percentage displacement of the PI3Kγ/PKA-RIIα complex by DRI-Pep #20 or PI3Kγ MP, calculated from steady-state emission spectra of the PI3Kγ/PKA-F5M complex in the presence of increasing concentrations of the peptides (0–5 μM). The displacement efficiency was expressed as percentage of the binding between PI3Kγ and PKA-F5M relative to that in the absence of peptides. G , cAMP concentrations in peritoneal macrophages from WT (in green ) and PI3Kγ −/− mice (in gray ) treated with DRI-Pep #20 (1–25 μM) for 30 min. The amount of cAMP was expressed as percentage of cAMP accumulation observed in untreated PI3Kγ −/− cells. n ≥ 6 technical replicates from N > 3 independent experiments. ∗∗∗ p < 0.001 WT versus PI3Kγ −/− and # p < 0.05, ## p < 0.01, and ### p < 0.001 UT versus DRI-Pep #20 by one-way ANOVA, followed by Bonferroni’s post hoc test. Data are means ± SD. AU, arbitrary units; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα; PI3Kγ, phosphoinositide 3-kinase gamma; PKA-F5M, fluorescein 5-maleimide–labeled PKA-RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Sequencing, Fluorescence, Spectroscopy, Recombinant, Labeling, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Binding kinetics of the interaction between DRI-Pep #20 or PI3Kγ MP and PKA-RIIα

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Structural prediction of the binding between DRI-Pep #20 and PKA-RIIα. A , DRI-Pep #20 structure prediction by PEP-FOLD3.5. P1-G and RHQGK domains are shown as cartoons in gray and red , respectively. R-1, H-2, Q-3, and K-5 residues are indicated and shown as sticks . B , circular dichroism spectra of DRI-Pep #20 showing a peak at 190–240 nm. The percentage of α-helical and β-sheet secondary structures calculated by the K2D3 software are indicated. C , molecular docking simulation of the interaction between DRI-Pep #20 and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of DRI-Pep #20 in complex with residues 2 to 44 of PKA-RIIα (cartoon in green ) is shown. The key residues involved in the binding are indicated and shown as sticks , with DRI-Pep #20 residues in bold . Hydrogen bonds between DRI-Pep #20 and PKA-RIIα are indicated by yellow dashed lines . In ( A and C ), the structural models were developed using PyMOL. DRI, D-retroinverso; HADDOCK, high ambiguity driven biomolecular DOCKing; PI3Kγ, phosphoinositide 3-kinase gamma; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Binding Assay, Circular Dichroism, Software

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Structural prediction of the native binding between the N-terminal domain of PI3Kγ and PKA-RIIα. A , molecular docking simulation of the interaction between PI3Kγ and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of residues 109 to 159 of PI3Kγ in complex with residues 2 to 44 of the PKA-RIIα dimer ( green cartoon) is shown. The amino acids critical for the binding between the two proteins are shown and indicated as sticks , with the residues of PI3Kγ in bold . The putative PKA-binding motif of PI3Kγ (126–150) is shown in orange and blue . The sequence in orange indicates the region of PI3Kγ that was identified as being at the core of the interaction (KATHR). Hydrogen bonds between PI3Kγ and PKA-RIIα are indicated by yellow dashed lines . B , structural prediction of the KATHR sequence by PEP-FOLD3.5. KATHR and P1-G domains are shown as cartoons in orange and gray , respectively. K-18, H-21 and R-22 residues of the KATHR sequence (corresponding to K-126, H-129 and R-130 of native PI3Kγ) are indicated and shown as sticks . C , molecular docking simulation of the interaction between KATHR and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of KATHR in complex with residues 2 to 44 of PKA-RIIα (cartoon in green ) is shown. Yellow dashed lines indicate hydrogen bonds between KATHR and 2 to 44 PKA-RIIα. The amino acids critical for the binding are indicated and shown as sticks , with KATHR residues in bold . Throughout, the structural models were developed using PyMOL. HADDOCK, high ambiguity driven biomolecular DOCKing; PI3Kγ, phosphoinositide 3-kinase gamma; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Binding Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: DRI-Pep #20 increases cAMP levels locally in vivo in the airway tract of mice. A , schematic representation of the treatment schedule. Mice received DRI-Pep #20 through intratracheal (i.t.) instillation. B – D , cAMP concentrations in tracheas ( B ), lungs ( C ) and hearts ( D ) from BALB/c mice 24 h after i.t. instillation of different doses of DRI-Pep #20 (0–750 mg/kg). Values in brackets indicate the dose of DRI-Pep #20 expressed as mg/kg. The number of mice (n) ranged from three to six per group. EC 50 , median effective concentration. E – G , cAMP concentrations in tracheas ( E ), lungs ( F ) and hearts ( G ) from WT and PI3Kγ −/− mice 24 h after i.t. instillation of 10 μg/Kg DRI-Pep #20 (in green ) or PBS (in gray ). The number of mice (n) ranged from three to four per group. In ( A and B ), ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 by one-way ANOVA, followed by Bonferroni’s post hoc test. In ( E and F ) ∗ p < 0.05 and ∗∗ p < 0.01 PBS versus DRI-Pep #20 by two-way ANOVA test, followed by Bonferroni’s post hoc analysis. Throughout, data are means ± SD. DRI, D-retroinverso; PI3Kγ, phosphoinositide 3-kinase γ.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: In Vivo, Concentration 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: 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