Journal: Journal of Clinical Neurology (Seoul, Korea)

Article Title: Education Moderates the Biomarker–Cortical-Thickness Relationship in Females With Cognitive Decline: A Descriptive Study With Sex Comparisons

doi: 10.3988/jcn.2025.0686

Figure Lengend Snippet: Group comparisons according to sex and education

Article Snippet: The plasma levels of amyloid-β 42 (Aβ42), phosphorylated tau 181 (p-tau), and total tau were quantified using the Single Molecule Array (SIMOA) platform (Quanterix), from which the p-tau/Aβ42 ratio was calculated.

Techniques:

Journal: Poultry Science

Article Title: Heterologous expression, immunogenic evaluation, and subunit vaccine potential of the σC protein from the Xinjiang avian reovirus (ARV) strain xj-1.1

doi: 10.1016/j.psj.2026.106779

Figure Lengend Snippet: Standard curve for phosphorylation level determination of recombinant σC proteins.

Article Snippet: Phosphorylation levels were quantified using the formula provided by the Phosphorylation Assay Kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: C500061): N (Protein phosphorylation level%) = (X × 2.24% × A)/(B × 31) Where: N: Molar percentage of phosphorus per mole of the target phosphorylated protein; A: Molecular weight of the target protein (g/mol); B: Total protein concentration of the sample (μg/mL); X: Phosphorylated protein concentration of the sample (μg/mL); Note: The phosphorylation level of the phosphorylated standard protein used in this assay was 2.24%.

Techniques: Phospho-proteomics, Recombinant

Journal: Molecules

Article Title: Discovery of a Novel Coumarin/Thiazole Chalcone Hybrid as a Potent Dual Inhibitor of Tubulin and Carbonic Anhydrases IX & XII with Promising Anti-Proliferative Activity

doi: 10.3390/molecules31060917

Figure Lengend Snippet: Effect of compound 6 on VEGF-stimulated VEGFR-2 (Tyr1175) phosphorylation in HUVEC cells.

Article Snippet: Phosphorylated VEGFR-2 (Tyr1175) levels were quantified using the PathScan ® RP Phospho-VEGFR-2 (Tyr1175) Sandwich ELISA Kit (#7335, Cell Signaling Technology, Danvers, MA, USA) following the rapid protocol.

Techniques: Phospho-proteomics

Journal: Journal of Nanobiotechnology

Article Title: ROS-responsive hydrogel-delivered miR-665 targets STAT3 to alleviate inflammation and promote hair follicle regeneration in alopecia areata

doi: 10.1186/s12951-026-04214-7

Figure Lengend Snippet: ( a ) Isolation and characterization of UMSC-EVs. ( b ) Isolation and characterization of EMSC-EVs. ( c ) Characterization of UMSC-EVs and EMSC-EVs by WB. ( d ) AA mouse model. ( e ) H&E staining of skin from AA mice. ( f ) H&E staining of skin from healthy mice. ( g ) Hair growth in each group on day 15. ( h ) Tracing results of EMSC-EVs (green fluorescence). ( i ) Venn diagram. ( j ) Volcano plot. k . Bubble plot of pathway enrichment analysis. l . miR-665 expression in UMSC-EVs and EMSC-EVs. m . Preliminary screening of miRNAs. n . Target prediction of miR-665 via miRDB database. o . Mechanism of miR-665 targeting STAT3 mRNA. p . The results of the dual-luciferase reporter assay. ( n = 3 per group; ns = not significant, *** P < 0.001, **** P < 0.0001)

Article Snippet: After transfer to polyvinylidene difluoride membranes, rabbit antibodies against phosphorylated STAT3 ( p -STAT3) (1∶2000, CST), mouse antibody against stat3 (1∶2000, CST), mouse antibody against β-actin (1∶1000, Beyotime), and mouse antibody against STAT3 (1∶1000, Beyotime) were used.

Techniques: Isolation, Staining, Fluorescence, Expressing, Luciferase, Reporter Assay

Journal: Journal of Nanobiotechnology

Article Title: ROS-responsive hydrogel-delivered miR-665 targets STAT3 to alleviate inflammation and promote hair follicle regeneration in alopecia areata

doi: 10.1186/s12951-026-04214-7

Figure Lengend Snippet: ( a ) STAT3 expression in lentivirus-transfected HaCaT cells and DPCs. ( b ) STAT3 expression in lentivirus-transfected HaCaT cells and DPCs before and after IFN-γ treatment. ( c ) STAT3 expression in IFN-γ-treated HaCaT cells and DPCs. ( d ) WB results showing STAT3 expression in lentivirus-transfected HaCaT cells before and after IFN-γ treatment. ( e ) WB results showing STAT3 expression in lentivirus-transfected DPCs before and after IFN-γ treatment. ( f ) Scratch assay results of HaCaT cells. ( g ) 48 h Transwell assay results of DPCs. ( h ) WB results of rescue experiments in HaCaT cells and DPCs. ( i ) Growth of hair follicles in ex vivo culture on day 5 under different treatment conditions. ( n = 3–6 per group; * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: After transfer to polyvinylidene difluoride membranes, rabbit antibodies against phosphorylated STAT3 ( p -STAT3) (1∶2000, CST), mouse antibody against stat3 (1∶2000, CST), mouse antibody against β-actin (1∶1000, Beyotime), and mouse antibody against STAT3 (1∶1000, Beyotime) were used.

Techniques: Expressing, Transfection, Wound Healing Assay, Transwell Assay, Ex Vivo

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A. Schematic representation of the CAR construct used in this study: a second-generation CAR receptor containing a CD28 hinge and transmembrane domain, a CD28 costimulatory domain, and CD3ζ signaling domain. B. Schematic representation of the protocol used to manufacture CAR-T cells. Healthy donor-derived PBMCs were activated using an anti-CD3 antibody. After 48h, expanded T cells were transduced with a retroviral vector to induce expression of the CAR. Transduction efficiency was assessed 5-7 days after transduction and cells were used for functional analysis between 7-14 days after transduction. C. Schematic representation of the protocol used to detect phosphorylation in CAR-T cells. CAR-T cells were cocultured with PSCA-expressing HPAC (HPAC WT ) or PSCA-deficient HPAC (HPAC PSCA-KO ) tumor cells for 1, 10, 30 and 60 minutes, followed by protein extraction. For cytoplasmic proteins (ZAP70, PLCγ, VAV1), phosphorylation was detected in the whole cell extract (WCE). For detection of phosphorylated CD28 Y218, the CAR was enriched through immunoprecipitation. D. Left Panel. Representative Western blot showing the phosphorylation of CD28 Y218, Zap70 Y319, PLCγ Y783, VAV1 Y174 in PSCA-specific CAR-T cells, following stimulation with HPAC WT or HPAC PSCA-KO cells. Right Panel. Phosphorylation intensity quantified by densitometry (representative plot). CD28 pY218, ZAP70 pY319, PLCγ pY783 and VAV1 pY174 normalized with respect to total CAR (CD3ζ), total ZAP70, total PLCγ and total VAV1 respectively. Untransduced cells (UT) were included as a negative control of immunoprecipitation. E. T cells were isolated from PBMCs and stimulated using CD3/CD28 T -activator Dynabeads®. CD28 was then enriched through immunoprecipitation for further analysis. Left Panel. Representative Western blot showing the phosphorylation of endogenous CD28 Y218 at the indicated time points after stimulation. Right Panel. Phosphorylation intensity quantified by densitometry. CD28 pY218 was normalized with respect to total CD28.

Article Snippet: To detect Y218 phosphorylation of endogenous CD28, T cells were isolated from healthy donor- derived PBMCs using the Pan T Cell Isolation Kit – human (Miltenyi Biotec, 130-096-535).

Techniques: Construct, Derivative Assay, Transduction, Retroviral, Plasmid Preparation, Expressing, Functional Assay, Phospho-proteomics, Protein Extraction, Immunoprecipitation, Western Blot, Negative Control, Isolation

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Schematic representation of WT and PYRP mutant CAR constructs. In the PYRP mutant, alanine (A) 217 and serine (S) 220 were substituted with prolines (P) to generate a proline-rich domain. B . PSCA-targeted WT and PYRP CAR-T cells were cocultured with HPAC WT cells for 0, 1, 10, 30, or 60 min. CAR molecules were immunoprecipitated using protein-L coated beads and co-immunoprecipitated proteins were analyzed by Western blot. Upper panel: Representative Western blot results showing ITK present in CAR immunoprecipitate. CD3ζ staining of the CAR molecule was used as a bait control. Lower panel: Intensity of ITK with respect a total CAR (CD3ζ) was analyzed by densitometry (representative plot). C. PSCA WT and PYRP CAR-T cells were stimulated with HPAC WT cells for 10 min and phosphorylation of Y218 was analyzed by mass spectrometry. Bar charts represent the intensity of phosphorylated Y218 normalized to the total CAR signal. Significance was determined by one-way Anova. * = P<0.05. Data is represented as the mean ± standard deviation (SD). D and F. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Significance was determined by one-way ANOVA. * = P<0.05, ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). E. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. RNA was extracted after 24 hours and IL-2 mRNA production was evaluated using quantitative Time PCR. CAR mRNA was used as normalization control. Significance was determined by one-way Anova. ** = P<0.01, *** = P<0.001. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). G. % of change in tumor volume (compared to baseline) is represented (n=5). Representative example of two independent experiments. Significance was determined by permutation two sample t-test. * = P<0.05.

Article Snippet: To detect Y218 phosphorylation of endogenous CD28, T cells were isolated from healthy donor- derived PBMCs using the Pan T Cell Isolation Kit – human (Miltenyi Biotec, 130-096-535).

Techniques: Mutagenesis, Construct, Immunoprecipitation, Western Blot, Staining, Control, Phospho-proteomics, Mass Spectrometry, Standard Deviation, Enzyme-linked Immunosorbent Assay