Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: TGF-β1 production of NK1.1-CD4+NKG2D+ cells upon various stimulations at different times. A. TGF-β1 of NK1.1-CD4+NKG2D+ cells detected by flow cytometry at 0.5-, 2-, 8-, 16-hour stimulation. B. Statistical analysis of TGF-β1 variations at different time points. C. TGF-β1 transcription of NK1.1-CD4+NKG2D+ cells detected by real-time PCR. D. Concentration of TGF-β1 in supernatants of NK1.1-CD4+NKG2D+ cells upon indicated stimulations measured by ELISA. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Activation of PI3K, MAPK, STAT3 and NF-κB signaling pathways. Western blot analysis of PI3K-p85, -p110, Akt, pAkt in NK1.1-CD4+NKG2D+ cells upon different stimulations at 2-hour (A) or 8-hour (C). Statistical analysis of expression variations of PI3K-p85, -p110, Akt, pAkt at 2-hour (B) or 8-hour (D). Analysis of JNK, Erk, p38 (E, F), NF-κB, STAT3 (G, H) and their phosphorylation in NK1.1-CD4+NKG2D+ cells upon stimulations for 8 hours. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Expressing, Phospho-proteomics

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of PI3K/JNK/AP-1 on TGF-β1 expression. NK1.1-CD4+NKG2D+ cells were treated with the PI3K inhibitor (LY294002) under different stimulations for 8 hours (A). (B) Statistical analysis of effects of LY294002 on TGF-β1 expression. (C, D) Effects on JNK/pJNK expression by the PI3K inhibitor (LY294002). Effects of JNK inhibitor (E, F), Erk or p38 inhibitor (G), and AP-1 inhibitor (H, I) on TGF-β1 expression of NK1.1-CD4+NKG2D+ cells. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of NF-κB on TGF-β1 expression. Effects of Bortezomib (NF-κB inhibitor) on TGF-β1 expression (A, B). Effects of LY294002 on NF-κB p65 activation in NK1.1-CD4+NKG2D+ cells for 8 hours (C, D). Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing, Activation Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of STAT3 on TGF-β1 expression. Effects of STAT3 inhibitors on TGF-β1 expression in NK1.1-CD4+NKG2D+ cells for 8 hours (A). (B, C) Effects of LY294002 on STAT3-pY705 phosphorylation. (D, E) Effects of Bortezomib on STAT3-pY705 phosphorylation. (F) The predicted binding sites of the TGF-β promoter by STAT3. (G) STAT3 engagement measured by a ChIP assay. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing, Phospho-proteomics, Binding Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Diagram of the TGF-β1 transcription in NK1.1-CD4+NKG2D+ cells regulated by AP-1, NF-κB, and STAT3.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques:

Journal: Neurochemical Research

Article Title: Morin Improves Cognitive Deficits in an in Vivo Model of Vascular Dementia by Modulating the N-methyl-D-aspartate Receptor Signaling Pathways

doi: 10.1007/s11064-026-04717-7

Figure Lengend Snippet: Morin modulated the expression of NMDA receptors in the hippocampus of VaD rats. a the expression levels of NR1 ; b the expression levels of NR2A ; c the expression levels of NR2B ; d the expression levels of NR1 protein; e the expression levels of NR2A protein; f the expression levels of NR2B protein; g protein levels of p-CREB; h protein levels of p-CAMK2A; i protein levels of p-CAMK2D. Protein levels of NR1, NR2A, and NR2B were quantified by ELISA. Data are presented as mean ± SD ( n = 8 per group). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (data met assumptions of normality and homoscedasticity)/Kruskal-Wallis with Dunn’s test (data did not meet assumptions). * indicates a significant difference from the Sham group; # indicates a significant difference 2VO group; *# indicates a significant difference from both the Sham and 2VO groups, with a p-value of less than 0.05 considered statistically significant

Article Snippet: Moreover, phosphorylation levels of calcium/calmodulin-dependent protein kinase II isoforms CAMK2A and CAMK2D at Thr286 (p-CAMK2A, p-CAMK2D) were quantified using the Colorimetric Cell-Based ELISA Kit (CAMK2A/CAMK2D (Phospho-Thr286), Boster Bio, #EKC2366).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Neurochemical Research

Article Title: Morin Improves Cognitive Deficits in an in Vivo Model of Vascular Dementia by Modulating the N-methyl-D-aspartate Receptor Signaling Pathways

doi: 10.1007/s11064-026-04717-7

Figure Lengend Snippet: Morin modulated the expression of NMDA receptors in the hippocampus of VaD rats. a the expression levels of NR1 ; b the expression levels of NR2A ; c the expression levels of NR2B ; d the expression levels of NR1 protein; e the expression levels of NR2A protein; f the expression levels of NR2B protein; g protein levels of p-CREB; h protein levels of p-CAMK2A; i protein levels of p-CAMK2D. Protein levels of NR1, NR2A, and NR2B were quantified by ELISA. Data are presented as mean ± SD ( n = 8 per group). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (data met assumptions of normality and homoscedasticity)/Kruskal-Wallis with Dunn’s test (data did not meet assumptions). * indicates a significant difference from the Sham group; # indicates a significant difference 2VO group; *# indicates a significant difference from both the Sham and 2VO groups, with a p-value of less than 0.05 considered statistically significant

Article Snippet: Moreover, phosphorylation levels of calcium/calmodulin-dependent protein kinase II isoforms CAMK2A and CAMK2D at Thr286 (p-CAMK2A, p-CAMK2D) were quantified using the Colorimetric Cell-Based ELISA Kit (CAMK2A/CAMK2D (Phospho-Thr286), Boster Bio, #EKC2366).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a Relative quantification of Kif5a, Kif5b and Kif5c transcripts by real-time PCR in CD8α + and CD11b + DCs and BMDCs from WT or cKO Kif5b mice. Transcript levels for each sample are expressed relative to Kif5b. Graph shows mean ± S.E.M. ( n = 3). b Contour plots of DCs from the spleen of either WT or cKO Kif5b mice pregated on CD19 − , Gr − , F4/80 − CD11c + , and IA/IE + . The data are representative of three independent experiments. c Absolute numbers of CD11b + DCs and CD8α + DCs in the spleen of WT (blue circles) and cKO Kif5b (red squares) mice. The data are representative of five independent experiments. d – g The efficiency of cross-presentation of different concentration of sOVA and the presentation of OVA peptide SIINFEKL in vitro by CD8α + DCs ( d , g ), CD11b + DCs ( e , g ) and BMDCs ( f , g ) from WT (blue histogram or line) or cKO Kif5b (red histogram or line) mice was measured as IL-2 secretion by OT-I T cells after 16 h of co-culture. Graph shows mean ± S.E.M. ( n = 4). Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. * P < 0.05; ** P < 0.005; *** P < 0.0001.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Quantitative Proteomics, Real-time Polymerase Chain Reaction, Concentration Assay, In Vitro, Co-Culture Assay, Comparison

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a Tumour growth curves for WT (blue line) or cKO Kif5b (orange line) mice injected subcutaneously with B16-OVA cells and adoptively transferred with OT-I T cells (WT purple line, cKO Kif5b yellow line). Tumour growth was monitored daily, and non-survival was defined as ulceration of the tumour or a mean tumour diameter of 15 mm. The data are quoted as the mean ± SEM from two independent experiments (WT, WT OT1, cKO Kif5b n = 10 mice per group, cKO Kif5b OT1 n = 9 mice). Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. * P < 0.05; ** P < 0.005; *** P < 0.0001. b Survival curves for WT or cKO Kif5b mice, treated as in a . Statistical significance was determined by the log-rank test and by the Gehan–Breslow–Wilcoxon test. * P < 0.05; ** P < 0.005. c Mice were injected with Violet-labelled transgenic OT-I T cells and primed 1 day later with CD11c/P3UOVA (WT blue line or circles, cKO Kif5b red line or circles; WT mice n = 7, cKO Kif5b mice n = 7). The left panel shows representative profiles gated on CD8 + , TCR Vα2 + cells. Statistical analysis of the division index is shown in the right panel: ** P < 0.005 in a two-tailed unpaired Student’s t test. Graph shows mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Injection, Comparison, Transgenic Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a CD8α + , CD11b + DCs and BMDCs from WT (blue histogram) or cKO Kif5b (red histogram) mice were incubated for 10, 30 or 60 min at 37 °C or for 10 min at 4 °C with Alexa-Flour-647-OVA prior to flow cytometry analysis. The figure shows a histogram that was representative of three independent experiments. b CD8α + , CD11b + DCs and BMDCs from WT (blue line) or cKO Kif5b (red line) mice were incubated for 30 min at 37 °C with DQ-OVA. Next, the cells were washed. DQ-OVA degradation was analysed by flow cytometry after different chase periods. Graphs are representative of four independent experiments. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. *** P < 0.0001. c Degradation of DQ-OVA in BMDCs from WT (DMSO blue line, Conc-A or Cath I purple line) or cKO Kif5b (DMSO orange line, Conc-A or Cath I yellow line) mice pretreated or not with concanamycin A (Conc-A) or a cathepsin inhibitor-I (Cath I). Graphs are representative of four independent experiments. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. ** P < 0.005; *** P < 0.0001. d The pH values in CD8α + , CD11b + DCs and BMDCs from WT (blue line) or cKO Kif5b (red line) mice were determined by FACS after a pulse and different chase periods. Graphs are representative of four independent experiments. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. * P < 0.05; ** P < 0.005; *** P < 0.0001. e Protease activity of CatB/L in early (20 min) or late (120 min) using total cell lysate from BMDCs from WT (blue histogram) or cKO Kif5b (red histogram) mice was measured with a specific fluorescent substrate. Graphs are representative of three independent experiments. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. *** P < 0.0001. a – e Graphs show mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Incubation, Flow Cytometry, Comparison, Activity Assay

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: BMDCs from WT or cKO Kif5b mice were incubated for 15, 30 or 60 min with Alexa-Flour-647-OVA and then plated on glass coverslips. The cells were fixed, permeabilized and stained with anti-EEA1, anti-Lamp1 or anti-Rab11 antibodies. The left panel shown representative images for the 15 min time point observed in three independent experiments. Bars: 5 μm. n = 32 cells per condition. Statistical significance (shown in the right panel; WT blue circle, cKO Kif5b red square) was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. ** P < 0.005; *** P < 0.0001. Graphs show mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Incubation, Staining, Comparison

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a CD8α + , CD11b + DCs and BMDCs from WT (blue line) or cKO Kif5b (red line) mice were labelled for H-2K b and analysed using FACS. The figure shows a FACS histogram that was representative of three independent experiments. b MHC-I recycling ability in CD8α + , CD11b + DCs and BMDCs from WT (blue line) or cKO Kif5b (red line) mice was measured using FACS at the indicated time points. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. ** P < 0.005; *** P < 0.0001. c TrfR (CD71) recycling ability was measured using FACS at the indicated time periods in CD8α + , CD11b + DCs and BMDCs from WT or cKO kif5b mice. Statistical analysis: * P < 0.05; ** P < 0.005; *** P < 0.0001 in the two-way ANOVA and Sidak test’s correction for multiple comparison. d MHC-I recycling in the presence of primaquine in BMDCs from WT or cKO Kif5b mice was measured using FACS at the indicated time points. Statistical analysis: ** P < 0.005; in the two-way ANOVA and Sidak test’s correction for multiple comparison. b – d Graphs are representative of four independent experiments. e BMDCs from WT or cKO kif5b mice were plated on glass coverslips and were fixed, permeabilized and stained with anti-EEA1, anti-Rab11 or anti-MHC-I antibodies. Representative images are shown in the left panel observed in three independent experiments. ( n = 33 cells per condition upper panel, n = 29 cells per condition lower panel) Bars: 5 μm. Statistical analysis (shown in the right panel; WT blue circle, cKO Kif5b red square): ** P < 0.005; *** P < 0.0001 in an two-tailed unpaired Student’s t test. b – e Graphs show mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Comparison, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a Confocal microscopy of BMDCs from WT or cKO Kif5b mice stained with anti-tubulin and anti-Kif5b. Bars: 5 μm. The indicated box is shown at a higher magnification in the inset. Bars: 2 μm. b Confocal microscopy of WT BMDCs stained with anti-EEA1 and anti-Kif5b. The intersection between the two stainings is shown in white. The indicated boxes are shown at higher magnification in the insets. Bars: 2 μm. c A schematic representation of early endosome WGA labelling. d WT and cKO Kif5b BMDCs were labelled with WGA-488 and stained with anti-EEA1. Bars: 2 μm (left panel). Quantification of the colocalization between WGA-488 and EEA1-555 (right panel; WT blue histogram, cKO Kif5b red histogram; n = 7 cells per condition). All images of single cells are representative of >100 cells observed in three independent experiments ( a – d ). e Spinning disk video microscopy was performed on WT and cKO Kif5b BMDCs labelled with WGA-488. Representative series of images are shown every 25 s. Bars: 2 μm. Images are representative of four independent experiments. See also Supplementary Movies and . f The number of tubulations unable to detach per cell during the 5 min acquisition. (WT blue circles, cKO Kif5b red squares; n = 20 cells per condition). Statistical analysis: *** P < 0.0001 in a two-tailed unpaired Student’s t test. g The number of WGA-488-positive vesicular structures >1 μm in size was measured in WT (blue line) and cKO Kif5b (red line) BMDCs during the 5 min acquisition ( n = 13 cells per condition). Statistical analysis: *** P < 0.0001 in a two-way ANOVA and Sidak test’s correction for multiple comparison. h Schematic representation of the WGA labelling protocol (upper panel). Spinning disk video microscopy was performed on WT and cKO Kif5b BMDCs labelled with WGA-555 and WGA-488. Representative series of images observed in three independent experiments are shown at the indicated time periods (lower panel). Bars: 2 μm. i n = 10 cells per condition. Statistical analysis of the experiment in (WT blue histogram, cKO Kif5b red histogram) ( h ); *** P < 0.0001 in a two-way ANOVA and Sidak test’s correction for multiple comparison. d – i Graphs show mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Confocal Microscopy, Staining, Microscopy, Two Tailed Test, Comparison

Journal: Frontiers in Pharmacology

Article Title: Salidroside derivative SHPL-49 enhances synaptic remodeling in BCCAO rats via the CDK5/p35/p25 signaling pathway

doi: 10.3389/fphar.2026.1727177

Figure Lengend Snippet: SHPL-49 promotes the expression of CDK5 at both mRNA and protein levels. (A,B) Correlation analysis among samples. (C,D) Differential expression analysis between experimental groups. (E) Venn diagram showing the overlap of differentially expressed genes. (F) GO enrichment analysis of differentially expressed genes. (G–J) qRT-PCR validation of selected differentially expressed genes in primary neurons. (K–N) qRT-PCR validation of differentially expressed genes in rat brain tissue. (O) Representative Western blot images of CDK5 expression in primary neurons. (P) Quantitative analysis of CDK5 protein levels in primary neurons. (Q) Representative Western blot images of CDK5 expression in rat brain tissue. (R) Quantitative analysis of CDK5 protein levels in rat brain tissue. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, OGD/R group vs. Control group, Model group vs. Sham group compared with the Control group, # P < 0.05, ## P < 0.01, ### P < 0.001, SHPL-49 group vs. OGD/R group. n = 3 for mRNA analysis in primary neurons and rat brain tissue. n = 6 for protein analysis in primary neurons and rat brain tissue.

Article Snippet: Membranes were blocked with 5% non-fat milk (Beyotime, China) in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C with the following primary antibodies: SYP (1:1,000, CST, United States), SYN1 (1:1,000, Boster, China), PSD95 (1:1,000, ABCAM, United States), CDK5 (1:1,000, Boster, China), p35/25 (1:1,000, CST, United States), p-PSD95 (1:1,000, CST, United States).

Techniques: Expressing, Quantitative Proteomics, Quantitative RT-PCR, Biomarker Discovery, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Salidroside derivative SHPL-49 enhances synaptic remodeling in BCCAO rats via the CDK5/p35/p25 signaling pathway

doi: 10.3389/fphar.2026.1727177

Figure Lengend Snippet: SHPL-49 inhibits the proteolytic conversion of p35 to p25. (A) Representative Western blot images showing p35 and p25 expression in primary neurons. (B) Quantitative analysis of p35 protein lev-els in primary neurons. (C) Quantitative analysis of p25 protein levels in primary neurons. (D) Representative Western blot images of p35 and p25 in rat brain tissue. (E) Quantitative analysis of p35 protein levels in rat brain tissue. (F) Quantitative analysis of p25 protein levels in rat brain tis-sue. (G) Representative Western blot images showing the expression levels of CDK5, p35, p25, and p-PSD95/PSD95 ratio in primary neurons following OGD/R treatment and co-treatment with SHPL-49 and CDK5 inhibitor Roscovitine. (H) Quantitative analysis of CDK5 protein levels in primary neurons under the same experimental conditions. (I) Quantitative analysis of p35 protein levels in primary neurons under the same experimental conditions. (J) Quantitative analysis of p25 protein levels in primary neurons under the same experimental conditions. (K) Quantitative analysis of p-PSD95/PSD95 ratio in primary neurons under the same experimental conditions. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, OGD/R group vs. Control group, Model group vs. Sham group, # P < 0.05, ## P < 0.01, ### P < 0.001, SHPL-49 group or PD151746 group vs. OGD/R group, SHPL-49 group or SAL group vs. Model group n = 6 per group.

Article Snippet: Membranes were blocked with 5% non-fat milk (Beyotime, China) in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C with the following primary antibodies: SYP (1:1,000, CST, United States), SYN1 (1:1,000, Boster, China), PSD95 (1:1,000, ABCAM, United States), CDK5 (1:1,000, Boster, China), p35/25 (1:1,000, CST, United States), p-PSD95 (1:1,000, CST, United States).

Techniques: Western Blot, Expressing, Control