Journal: Molecular Neurobiology

Article Title: Ptbp2 Alleviates Neuroinflammation and Blood–brain Barrier Disruption via Modulating Microglial Polarization in Ischemic Stroke

doi: 10.1007/s12035-026-05704-3

Figure Lengend Snippet: Ptbp2 modifies microglial polarization by downregulating the NF-κB pathway ( a- e ) Representative western blots of p-p65 and p65 and quantitative analysis of protein levels normalized to GAPDH ( n = 3). ( e-f ) Quantitative analysis of TNF-α levels normalized to GAPDH and representative western blots ( n = 3). ( g-l ) ELISA for evaluating TNF-α and IL-10 levels in the mouse blood, brain tissue, and BV-2 cells ( n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: TNF-α and IL-10 concentrations were determined using the quick ELISA kits (TNF-α, BOSTER EK0527; IL-10, BOSTER EK0417) following the supplier's instructions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: Endogenous interleukin-22 prevents cardiac rupture after myocardial infarction in mice

doi: 10.1371/journal.pone.0286907

Figure Lengend Snippet: ( A ) Total lysates were prepared from whole ventricle before MI or infarct area 3 days after MI in WT and IL-22KO mice. Representative images are shown for Western blot for IL-22R1, IL-10RB and GAPDH. Graphs show the ratio of IL-22R1 or IL-10RB to GAPDH (n = 12 for each group); *P < 0.05 vs. Pre-MI, Kruskal-Wallis test/Dunn’s multiple comparison test. ( B ) Representative images and quantitative analysis of immunostaining for IL-22R1 in the hearts from WT mice before and 3 days after MI. Values are expressed as the percentage of IL-22R1-positive area per tissue area (n = 3–4 for each group); *P < 0.05 vs. Pre-MI, Mann-Whitney test. ( C ) Total lysates were prepared from whole ventricle before MI or infarct area 3 days after MI. Representative images are shown for the Western blots of p-STAT3, STAT3 and GAPDH. Graphs show the ratio of p-STAT3 to STAT3 (n = 10 for each group); ***P < 0.001, N.S., non-significant, one-way ANOVA/Dunnett’s T3 multiple comparison test.

Article Snippet: Antibody against IL-10RB (15102-1-AP, 450ng/mL) was purchased from Proteintech (Chicago, IL).

Techniques: Western Blot, Comparison, Immunostaining, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2 , and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH . (B) Effect of different maturation stimuli (CpG and LPS + IFN-γ or Poly I:C) on IL22RA2, IL12B , and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB . Extent of maturation is represented as percentage of CD83 + fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB was measured by RT-qPCR using pre-designed SYBR Green primers from the same cells. Representative experiment out of three performed. All primers used are listed in Supplementary Table .

Article Snippet: For confocal immmuofluorescence microcroscopy, we used the following antibodies: anti-IL22RA2 (R&D; AF1087 and BAF1087, 1:20 each), anti-IL22RA2 (Proteintech; 66190-1-Ig), anti-TGOLN (Atlas; HPA012723-100ul, 1:500), anti-ERp72 (Enzo; ADI-SPS-720-F; 1:500), donkey anti-goat A647 (Jackson ImmunoResearch; 705-605-147; 1:500), Streptavidin DyLight 488 (Tebu-bio; 039S000-41; 1:4000), donkey anti-rabbit A647 (Jackson Immunoresearch; 711-605-152; 1:500), as well as DAPI (Sigma; 10 μg/ml).

Techniques: Derivative Assay, Binding Assay, Variant Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, SYBR Green Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Differential secretion, intracellular retention and degradation by the proteasome of IL-22BP isoforms. Data refer to HEK293 cells transiently transfected for 24 h unless otherwise specified. (A) HEK293 cells were transiently transfected with expression vectors encoding IL-22BPi1, BPi2, or BPi3. Intracellular and secreted IL-22BP protein levels were measured by WB (FLAG Ab) and ELISA, respectively. Transfection efficiency was measured by IL22RA2 RT-qPCR relative to the housekeeping gene GAPDH (mean ± SEM; n = 3). (B) Confocal microscopy of IL-22BP isoforms (green, IL-22BP Ab 4 with either ERp72 or trans-Golgi marker TGOLN2 (red) in transfected HEK293 cells; cells were counterstained with DAPI. (C) Secreted recombinant IL-22BPi2 is not detected by WB in unconcentrated conditioned media (CM). Various cytokine expression vectors, as indicated, were transiently transfected and CM were collected and immunoblotted for FLAG. (D) Detection of IL-22BP isoforms in cell lysates (CL) and acetone-precipitated CM (AP) by FLAG Ab. Secreted IL-22BPi2 was treated with PNGase F or Endo H. (E) Intracelullar isoforms of IL-22BP were treated with PNGase F or Endo H and detected by FLAG Ab. (F) HEK293 cells were transfected for 24 h with expression vectors for either IL-2 or IL-2EX4. The latter was generated by subcloning exon-4 from IL22RA2v1 into the open reading frame of IL-2 through its unique Xba1 position. Detection of resulting proteins in AP, CL, CM by FLAG Ab. (G) HEK293 cells were transiently transfected with the same expression plasmids as in (A) or IL-2EX4; 24 h later, cells were treated with a mix of proteasome inhibitors (PI) (5 μM lactacystin, 5 μM MG132, and 1 mM epoxomicin), and 18 h later cells were lysed and immunoblotted for FLAG using GAPDH as a loading control. All data are from at least 3 independent experiments.

Article Snippet: For confocal immmuofluorescence microcroscopy, we used the following antibodies: anti-IL22RA2 (R&D; AF1087 and BAF1087, 1:20 each), anti-IL22RA2 (Proteintech; 66190-1-Ig), anti-TGOLN (Atlas; HPA012723-100ul, 1:500), anti-ERp72 (Enzo; ADI-SPS-720-F; 1:500), donkey anti-goat A647 (Jackson ImmunoResearch; 705-605-147; 1:500), Streptavidin DyLight 488 (Tebu-bio; 039S000-41; 1:4000), donkey anti-rabbit A647 (Jackson Immunoresearch; 711-605-152; 1:500), as well as DAPI (Sigma; 10 μg/ml).

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Confocal Microscopy, Marker, Recombinant, Generated, Subcloning

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Silencing of IL22RA2 in immature moDCs reduces GRP78 expression. (A) CD14 + monocytes were stained with DAPI (blue), trans-GOLGI (red) and IL-22BP (green, IL-22BP Ab 4). (B) Overall (best coverage, BC) and isoform-specific IL22RA2 expression levels in classical, intermediate and non-classical monocyte subpopulations (top) from four healthy donors (average ± SEM). Percentages of IL-22BP + monocytes found in each subpopulation by immunofluorescence microscopy (bottom) (mean ± SEM; n = 4; * p < 0.05, Kruskal-Wallis test for comparison of differences in IL22RA2 mRNA levels or IL-22BP + counts in the three subpopulations). (C) IL22RA2 expression levels in classical and intermediate immature moDCs at day 6 of cultivation. moDCs derived from non-classical monocytes showed high levels of cell death and had not experienced any changes in the shape of the cells. High Ct values of HK gene HPRT1 in qPCR compromised correct interpretation of IL22RA2 levels in this subgroup, which was therefore excluded from the graph. (D) Fold change relative to non-targeting siRNA control of IL22RA2 expression in CD14 + monocyte.-derived immature DCs on day 5 of cultivation following IL22RA2 silencing with Viromer Green at two concentrations of siRNA. (E) Silencing as described in (D) was not variant specific and both IL22RA2v1 and v2 were silenced to similar extent. (F) Scheme comparing efficiencies of four distinct silencing strategies on suppression of IL22RA2 mRNA (qPCR) and intracellular IL-22BP (ELISA) in immature CD14 + moDCs using 100 nM IL22RA2 siRNA or non-targeting siRNA in combination with Viromer Green. S, silencing, H, harvest. Bar diagrams represent fold change relative to non-targeting siRNA. (G) Effect of IL22RA2 silencing on GRP78 and ERp44 mRNA levels in CD14 + monocyte-derived immature DCs from healthy donors using silencing strategy B in (F) . CD14 + monocytes were cultivated in DM medium in the absence or presence of AM580 starting on day 0 up to the day of harvest. Average of fold change (±SEM) relative to non-silencing control over 6 (control) and 5 (AM580) independent measurements relative to the housekeeping gene HPRT1. * p < 0.03 by Wilcoxon test.

Article Snippet: For confocal immmuofluorescence microcroscopy, we used the following antibodies: anti-IL22RA2 (R&D; AF1087 and BAF1087, 1:20 each), anti-IL22RA2 (Proteintech; 66190-1-Ig), anti-TGOLN (Atlas; HPA012723-100ul, 1:500), anti-ERp72 (Enzo; ADI-SPS-720-F; 1:500), donkey anti-goat A647 (Jackson ImmunoResearch; 705-605-147; 1:500), Streptavidin DyLight 488 (Tebu-bio; 039S000-41; 1:4000), donkey anti-rabbit A647 (Jackson Immunoresearch; 711-605-152; 1:500), as well as DAPI (Sigma; 10 μg/ml).

Techniques: Expressing, Staining, Immunofluorescence, Microscopy, Derivative Assay, Variant Assay, Enzyme-linked Immunosorbent Assay