Journal: Inflammation

Article Title: Red Blood Cell-Derived Exosomes Deliver Complement C5 to Exacerbate Neuroinflammation and Neuronal Injury after Intracerebral Hemorrhage

doi: 10.1007/s10753-026-02456-z

Figure Lengend Snippet: ICH-Exos increased necroptosis and M1 polarization in hemin-induced primary murine microglia. ( A ) Cell viability was determined using the CCK-8 assay. ( B ) Cell apoptosis was assessed via flow cytometry. ( C ) Western blot analysis was conducted to measure p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL levels. ( D and E ) Flow cytometry was used to examine the proportions of CD86 + and CD163 + macrophages. ( F ) qRT-PCR analysis of iNOS, COX-2, CCL2, ARG1, CD206, and YM1 levels. ( G ) iNOS, COX-2, ARG1, and CD206 proteins levels. ( H ) ELISA was conducted to assess IL-1β, IL-6, and TNF-α levels. n = 3. * p < 0.05 vs. Control group; # p < 0.05 vs. Hemin group

Article Snippet: RIPK1 , 17519-1-AP , Rabbit , 1:1500 , 70–80 KDa , Proteintech , China.

Techniques: CCK-8 Assay, Flow Cytometry, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: Inflammation

Article Title: Red Blood Cell-Derived Exosomes Deliver Complement C5 to Exacerbate Neuroinflammation and Neuronal Injury after Intracerebral Hemorrhage

doi: 10.1007/s10753-026-02456-z

Figure Lengend Snippet: ICH-Exos increased necroptosis of macrophages/microglia and M1 polarization after ICH. ( A ) Cell apoptosis was detected by TUNEL. ( B ) p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL protein levels. ( C and D ) Immunofluorescence was used to detect IBA1 + iNOS + and IBA1 + ARG1 + . ( E ) iNOS, COX-2, CCL2, ARG1, CD206, and YM1 mRNA levels. ( F ) iNOS, COX-2, ARG1, and CD206 levels were analyzed by western blot. ( G ) ELISA was conducted to examine IL-1β, IL-6, and TNF-α. n = 5. * p < 0.05 vs. Sham group; # p < 0.05 vs. ICH group

Article Snippet: RIPK1 , 17519-1-AP , Rabbit , 1:1500 , 70–80 KDa , Proteintech , China.

Techniques: TUNEL Assay, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: Red Blood Cell-Derived Exosomes Deliver Complement C5 to Exacerbate Neuroinflammation and Neuronal Injury after Intracerebral Hemorrhage

doi: 10.1007/s10753-026-02456-z

Figure Lengend Snippet: ICH-Exos C5 promoted necroptosis and M1 polarization in hemin-induced microglia. ( A ) The heat map shows the differentially expressed proteins. ( B ) C5a level in brain tissue analyzed by IHC. * p < 0.05 vs. sham group. ( C ) C5a level in serum was detected by ELISA. * p < 0.05 vs. sham group. Under RIPA lysis conditions, the results of Western blot for C5a in ( D ) human RBC-Exos and ( E ) murine RBC-Exos after untreated EVs or after treatment with proteinase K (5 U/mL, 10 min) . # p < 0.05 vs. Sham-Exos/Normal-Exos group; @ p < 0.05 vs. ICH-Exos group. ( F ) C5a level in HMC3 cells was analyzed by Western blot and ELISA. ( G ) Cell viability was detected by CCK-8. ( H ) Cell apoptosis was detected by flow cytometry. ( I ) p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL levels were detected by Western blot. ( G ) The proportions of CD86 + M1 and CD163 + M2 macrophages were detected by flow cytometry. ( K ) iNOS, COX-2, CCL2, ARG1, CD206, and YM1 levels analyzed by qRT-PCR. ( L ) iNOS, COX-2, ARG1 and CD206 levels analyzed by Western blot. (M) ELISA was used to detect IL-1β, IL-6, and TNF-α levels. n = 3. # p < 0.05 vs. Normal-Exos group; & p < 0.05 vs. ICH-Exos group

Article Snippet: RIPK1 , 17519-1-AP , Rabbit , 1:1500 , 70–80 KDa , Proteintech , China.

Techniques: Paraffin-embedded Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Lysis, Western Blot, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR

Journal: Open Biology

Article Title: Manipulation of PK-M mutually exclusive alternative splicing by antisense oligonucleotides

doi: 10.1098/rsob.120133

Figure Lengend Snippet: Effects of ASOs on glioblastoma cells. ( a , b ) Exon 10 ASOs induce apoptosis in glioblastoma cells. ( a ) U87-MG or A172 cells were transfected with the indicated ASOs at a nominal final concentration of 90 nM, stained with Annexin V-APC/7-AAD 36 h after transfection and analysed by flow cytometry. The percentage of Annexin-V-positive cells is indicated for the two right quadrants in each plot. Each plot is a representative of three biological replicates. ( b ) ASO-induced apoptosis is dose-dependent. The indicated cells were transfected with ASOs 10W 45–59 or 10M 46–60 at 30, 60, or 90 nM, or ASO 10MS 139–153 at 60 or 90 nM. The control (Ctl) ASO was transfected at 90 nM. The percentage of Annexin-V-positive cells is plotted for each condition. The identity and dose of ASOs are indicated below the x -axis. Error bars represent s.d. ( n = 3). ( c , d ) Role of PK-M1 protein isoform in apoptosis induction. ( c ) Immunoblot analysis of A172 cells stably transduced with rtTA and doxycycline (dox)-inducible human T7-tagged PK-M1 cDNA. Cells were grown in parallel, with or without doxycycline, and harvested after 72 h. Antibodies used are indicated on the left. ( d ) Cells were grown as in ( c ) and then transfected with the indicated ASOs at 60 nM. Cells were then stained and analysed for Annexin V 36 h after transfection. Histograms indicate the percentage of Annexin-V-positive cells for each condition. Doxcycyline on/off conditions are indicated on the left. Error bars represent s.d. ( n = 3). ( e , f ) Role of PK-M2 protein isoform in apoptosis induction. A172 or U87 cells were stably transduced with T7-tagged human PK-M2 cDNA; transductants and the parental cell lines were transfected with the indicated ASOs at a nominal final concentration of 90 nM. ( e ) Immunoblot analysis of cells transfected with the indicated ASOs. Antibodies used are indicated on the left. ( f ) ASO-transfected cells were analysed for Annexin V as in ( d ). Histograms indicate the fold increase in Annexin-V-positive cells, compared with control ASO, for each cell line. Error bars represent s.d. ( n = 3).

Article Snippet: Primary antibodies were: β-actin (Genscript, mAb, 1 : 10 000); PK-M2 (Cell Signaling Technology, rAb, 1 : 2000); PARP (Cell Signaling Technology, rAb, 1 : 1000); T7 (mAb, 1 : 1000) and PK-M1 (ProteinTech, rAb, 1 : 1000).

Techniques: Transfection, Concentration Assay, Staining, Flow Cytometry, Control, Western Blot, Stable Transfection, Transduction

Journal: The Journal of Experimental Medicine

Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes

doi: 10.1084/jem.20242474

Figure Lengend Snippet: Intracellular portion of FCRL3 interacts with UBASH3A. (A) Schematic representation of the Protein A-TurboID experimental workflow. An anti-FCRL3 antibody was added to FCRL3-expressing or nonexpressing Jurkat cells followed by the addition of Protein A-TurboID fusion protein and biotin. After biotinylation and extensive washing, biotinylated proteins were recovered using streptavidin-conjugated beads and subjected to mass spectrometry analysis. (B) Example of Protein A-TurboID experiment. After incubation of FCRL3-expressing Jurkat cells (or empty vector control cells) with a mouse monoclonal anti-FCRL3 antibody together with recombinant Protein A-TurboID, biotinylated proteins were recovered by streptavidin pull-down, followed by western blot to confirm enrichment of FCRL3. The antibody used for immunoblot was a rabbit polyclonal anti-FCRL3. (C) Differentially retrieved proteins following FCRL3 Protein A-TurboID and mass spectrometry of FCRL3-expressing versus nonexpressing cells (log 2 FC ≥ |2|; P ≤0.01; N = 5 replicates). (D) Example of FCRL3 IP. Jurkat cells expressing FCRL3 (or empty vector control) were lysed, and 1.5 mg of protein extract was used for IP with a rabbit polyclonal anti-FCRL3 antibody, followed by western blot with the same antibody. Two independent representative FCRL3 IPs are shown. (E) Differentially retrieved proteins following FCRL3 IP-MS of FCRL3-expressing versus nonexpressing cells (log 2 FC ≥ |2|; P ≤0.01; N = 4 replicates). (F) Co-IP of FCRL3 with UBASH3A. HEK cells were transfected with the indicated plasmids, followed by IP of FCRL3 using a rabbit polyclonal anti-FCRL3 antibody and immunoblot for either UBASH3A (top) or FCRL3 itself (bottom). Data are representative of N = 2 independent experiments. (G) Schematic representation of the FCRL3 protein and C-terminal truncations (top). All proteins were efficiently expressed, as shown by western blot (bottom). (H) Heatmap showing the differentially enriched proteins by IP-MS of Jurkat cells transduced with full-length FCRL3 and its truncations. N = 3–4 independent samples. Significant differences between multiple experimental conditions were assessed with an ANOVA multiple-sample test (0.01 permutation-based FDR cut-off, 250 randomizations). Significant proteins were filtered, and Z-score normalization was applied to each protein across all samples. Unsupervised hierarchical clustering was employed to visualize on a heatmap proteins with a positive Z-score value for all replicates of the conditions LV-FCRL3 and LV-FCRL3-93 aa. IP, immunoprecipitation. Data underlying this figure can be found in . Source data are available for this figure: .

Article Snippet: The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.

Techniques: Expressing, Mass Spectrometry, Incubation, Plasmid Preparation, Control, Recombinant, Western Blot, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Transduction, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes

doi: 10.1084/jem.20242474

Figure Lengend Snippet: IP-MS and TurboID-MS confirm the interaction between FCRL3 and UBASH3A. (A) Schematic representation of FCRL3 and the C-terminally tagged FCRL3-3xFLAG protein. The 3xFLAG tag is shown as green squares. (B) Surface (left) FCRL3 staining and intracellular (right) anti-FLAG staining showing surface expression of the FCRL3-3xFLAG construct. (C) Western blot showing efficient immunoprecipitation of FCRL3-3xFLAG with anti-FLAG agarose beads in Jurkat cells expressing LV-FCRL3 and LV-FCRL3-3xFLAG. Immunoblot was performed using an anti-FCRL3 antibody. (D) Scatter plot showing the enriched proteins in the IP-MS of the FCRL3-3xFLAG compared with untagged FCRL3. In this experiment, to enhance the separation of true interactors from experimental noise, an additional filter was applied by selecting proteins present in at least 3 out of 4 replicates of the FCRL3-3xFLAG condition and in <2 replicates of the untagged control (FCRL3), followed by imputation of the missing values. A two-sided two-samples t test (0.01 permutation-based FDR cut-off, 250 randomizations) was employed to identify significant changes, and the results were visualized with a volcano plot generated with R, version 4.4.2. (E) Schematic representation of the Protein A-TurboID experimental workflow for UBASH3A. The anti-UBASH3A antibody was added to Jurkat cells ectopically expressing FCRL3 or the EGFR-FCRL3 140 aa chimera (or control cells), followed by the addition of Protein A-TurboID fusion protein and biotin. After streptavidin pull-down, biotinylated proteins were analyzed by mass spectrometry. (F) Differentially enriched proteins in the FCRL3-transduced cells versus control (left) and chimera-transduced cells versus control (right). N = 4 independent samples. Source data are available for this figure: .

Article Snippet: The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.

Techniques: Protein-Protein interactions, Staining, Expressing, Construct, Western Blot, Immunoprecipitation, Control, Generated, Mass Spectrometry

Journal: BioMed research international

Article Title: Effects of Hypoxic Environment on Periodontal Tissue through the ROS/TXNIP/NLRP3 Inflammasome Pathway.

doi: 10.1155/2022/7690960

Figure Lengend Snippet: Figure 2: Effects of systemic hypoxia stimulation on the expression of TXNIP/NLRP3 signaling pathway-related factors in periodontal tissues of rats: (a) immunofluorescence staining (IF) showed that TXNIP, NLRP3, ASC, and caspase-1 in periodontal tissues. Ab: alveolar; PL: periodontal ligament; R: root. Scale bars = 25 μm. (b) RT-PCR analysis of TXNIP, NLRP3, ASC, caspase-1, and IL-1β in periodontal tissues (n = 5). (c) Western blotting analysis of TXNIP, NLRP3, ASC, caspase-1, and IL-1β in periodontal tissues (n = 5 for each group). The above data are presented as the mean ± SEM, ∗p < 0:05.

Article Snippet: For IF, the sections were incubated overnight at 4°Cwith primary antibodies (1 : 250) diluted in PBS: TXNIP (Rabbit IgG, Boster, China), NLRP3 (Rabbit IgG, Boster, China), ASC (Rabbit IgG, Immunoway, USA), and caspase-1 (Rabbit IgG, Immunoway, China) and then incubated with Alexa Fluor-488 and Alexa Fluor-555 conjugate secondary antibodies at room temperature for 1h.

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot