Journal: The Journal of Biological Chemistry

Article Title: A tandem recruitment site in the pseudokinase scaffold PEAK3 is subject to phosphorylation-dependent regulation and cancer-associated mutations

doi: 10.1016/j.jbc.2026.111365

Figure Lengend Snippet: Serine/threonine protein kinases regulating PEAK3 S69 phosphorylation. A , consensus motifs of serine/threonine protein kinases with selectivity corresponding to the PEAK3 S69 region. Phosphorylation sites (position 0) are highlighted in pink and key features highlighted are arginine or lysine at position −3 ( orange ) and hydrophobic residues (ϕ) at +1 and −5 position ( blue ). X = any amino acid. Motifs sourced from Johnson et al. and PhosphoSitePlus ( , ). B , role of PKCs. MCF-10A/PEAK3 cells were treated with Go6850 or Go6976 and cell lysates subjected to Western blotting as indicated. Results are representative of four independent experiments. Histogram indicates the relative level of PEAK3 p-S69 normalized for HA and expressed relative to the DMSO control, which was arbitrarily set to 1.0. Data represent mean ± S.E.M., with 'au' indicating arbitrary units. ∗ p < 0.05, ∗∗ p < 0.01, by ratio paired t test. C , role of CaMKII as determined by pharmacological inhibition. MCF-10A/PEAK3 cells were treated with RA306 or vehicle control and cell lysates analyzed by Western blotting as indicated. Data are representative of three independent experiments. Results in C are derived from the same experiment and use the same DMSO control. Histogram indicates the relative level of PEAK3 p-S69 as mean ± S.E.M., ∗∗ p < 0.01, by ratio paired t test. D , role of CaMKII as determined by knockdown. CaMKIID and G were subject to siRNA-mediated knockdown (KD) in MCF-10A/PEAK3 cells and PEAK3 p-S69 assayed by Western blotting as indicated. Data are representative of three independent experiments. Histogram indicates the relative level of PEAK3 p-S69 as mean ± S.E.M. over three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, by ratio paired t test.

Article Snippet: The following reagents/inhibitors were used in this study: lambda protein phosphatase (New England Biolabs, P0753S), animal-free recombinant human EGF (PeproTech, AF-100–15), insulin from bovine pancreas (Merck, I-1882), CaMKII inhibitor RA306 (custom synthesized by Reagency, RGNCY-0117, 1 μM final), PKA Inhibitor 14 to 22 Amide, Cell-Permeable, Myristoylated (Calbiochem, 476,485, 10 μM final), DMSO (Sigma, d8418, 0.1% final), and MedChemExpress-sourced Akt inhibitor MK-2206 dihydrochloride (HY-10358, 100 nM final) and trametinib (HY-10999, 10 nM final).

Techniques: Phospho-proteomics, Western Blot, Control, Inhibition, Derivative Assay, Knockdown

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: Scientific Reports

Article Title: α-Synuclein oligomers mediate the aberrant form of spike-induced calcium release from IP 3 receptor

doi: 10.1038/s41598-019-52135-3

Figure Lengend Snippet: α-Synuclein oligomers suppressed CaBP1-induced inactivation of IP R and triggered spike-induced CICR from IP R. A. ( a ) Specimen recordings of I AHP in neurons with αSN or αSNo under the injection of CaBP1 Ab or CaBP1. Scale bars, 500 ms and 20 pA. ( b ) Summary diagram demonstrating average I AHP charge under the infusion of CaBP1 Ab, CaBP1, calmodulin (CaM) Ab, or CaM, each with αSN or αSNo. * p < 0.01, ** p < 0.02 (αSN vs αSNo, t -tests), + p < 0.01, ++ p < 0.02 (CaBP1 Ab vs no drug, αSN, t -tests) ( c ). Specimen recordings of action po t entials during positive current pulse in neurons with αSN or αSNo under the injection of CaBP1 Ab or CaBP1. Scale bars, 100 ms and 10 mV. ( d ) Average spike frequency during current steps under the infusion of CaBP1 Ab, CaBP1, CaM Ab, or CaM, each with αSN or αSNo. For the ‘no drug’ example in ( b , d ), the same data as shown in Fig. are reproduced for clarity. * p < 0.01, ** p < 0.02 (αSNo vs αSN, t -tests), + p < 0.01 (CaBP1 Ab vs no drug, αSN, t -tests).

Article Snippet: The pipette solution also included heparin (low molecular weight; 4 mg/ml; MP Biomedicals), ruthenium red, D-IP 3 , L-IP 3 (100 μM; Alomone Labs), glutathione S-transferase (GST)-CaBP1 (16 nM; Abnova Corp.), calmodulin (3 μM; BioVision Inc.), CaBP1 antibody (10 μg/ml; Novus Biologicals), and calmodulin antibody (10 μg/ml; Novus Biologicals).

Techniques: Injection

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions.

doi: 10.4049/jimmunol.174.10.6399

Figure Lengend Snippet: FIGURE 4. Adhesion of monocytes and MM6 cells to recombinant ad- hesion molecules. Leukocytes were perfused over plates coated with re- combinant CAM (f) or CAM plus recombinant HBP () for 4 min and the number of adherent cells was subsequently counted. Addition of recombi- nant HBP caused significantly increased binding of monocytes (A) (n 3 experiments) and MM6 cells (B) (n 4 experiments) to VCAM-1, P- selectin, and E-selectin but not to ICAM-1 and BSA. Values are presented as mean SD. , Significant difference (p 0.01).

Article Snippet: Plates coated with CAM (P-selectin, E-selectin, VCAM-1, ICAM-1) and recombinant HBP were produced by adding 50 l of recombinant human CAM (1 g/ml; R&D Systems) to the center of a well in a six-well plate.

Techniques: Recombinant, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions.

doi: 10.4049/jimmunol.174.10.6399

Figure Lengend Snippet: FIGURE 5. Intracellular Ca2 mobilization in MM6 cells after stimu- lation with recombinant HBP. A, Dynamic change in fluorescence intensity of MM6 cells loaded with fluo-4-AM after stimulation with recombinant HBP 500 ng/ml (E) or sham treatment (F). Measurements were made by flow cytometry in MM6 cell suspensions before and immediately after stimulation, and then at 30-s intervals. Values are expressed as a percent- age of MFI before treatment. , Significant difference (p 0.05) from sham treatment value (n 5). Values are presented as mean SD. B, Fluorescence intensity of fluo-4-loaded MM6 cells added to wells coated with individual CAM (f) or combinations of CAM and recombinant HBP (). Measurements were made in a fluorescence plate reader. Fluorescence intensity of MM6 cells in BSA-coated wells was considered as background fluorescence and subtracted from MFI of CAM-coated wells. , Significant difference (p 0.05) from MFI in the absence of recombinant HBP (n 6). Values represent mean SD. C, Dynamic changes in fluorescence intensity of individual MM6 cells loaded with fluo-4-AM after contact with the coated surfaces had been established. Fluorescent images were captured at 30, 60, and 90s after injection. Images are representative of three indepen- dent experiments with three wells in each experiment. Bars indicate 20 m.

Article Snippet: Plates coated with CAM (P-selectin, E-selectin, VCAM-1, ICAM-1) and recombinant HBP were produced by adding 50 l of recombinant human CAM (1 g/ml; R&D Systems) to the center of a well in a six-well plate.

Techniques: Recombinant, Cytometry, Fluorescence, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions.

doi: 10.4049/jimmunol.174.10.6399

Figure Lengend Snippet: FIGURE 6. Effect of intracellular calcium chelator BAPTA-AM on ar- rest of MM6 cells on recombinant adhesion molecules or on LPS-stimu- lated BAEC. MM6 cells were perfused over CAM-coated plates (A) or BAEC monolayers (B) for 4 min in the absence or presence of recombinant HBP and arrested cells were subsequently counted. Pretreatment of MM6 cells with BAPTA-AM consistently prevented the increase in adhesion evoked by recombinant HBP on all substrates. Values are presented as mean SD. , Significant difference (p 0.01). ns, Not significant.

Article Snippet: Plates coated with CAM (P-selectin, E-selectin, VCAM-1, ICAM-1) and recombinant HBP were produced by adding 50 l of recombinant human CAM (1 g/ml; R&D Systems) to the center of a well in a six-well plate.

Techniques: Recombinant

Journal: Nature communications

Article Title: LGALS3BP regulates centriole biogenesis and centrosome hypertrophy in cancer cells.

doi: 10.1038/ncomms2517

Figure Lengend Snippet: Figure 1 | A centrosomal protein-interaction network identifies new centrosomal proteins. (a) Protein–protein interaction subnetwork derived from the complete TAP interaction network (Supplementary Data 1) that displays known, newly identified or newly localized centrosome proteins (LGALS3BP and MAGED2). Interactions are represented as edges between bait proteins and their interaction partners. The bait proteins are displayed as hexagons, centrosomal proteins are coloured in grey and centriolar proteins in red. High-confidence interactions (Mascot score of the prey protein 450, at least two identified peptides) are shown with solid lines, and candidate interactions (Mascot score 424) are shown with dotted lines. (b,c) The protein–protein interactions of the main targets were validated by either reverse TAP (b) or co-immunoprecipitation (c). (b) The interactions of NME7 with TUBG1, TUBGCP2 and TUBGCP3 were confirmed by reverse TAP. The bait protein NME7 is identified through its TAP-tag moiety calmodulin- binding site (CBS). The cell extract was used as a positive western blotting control for the identification of the three NME7-interacting proteins. (c) The interaction between LGALS3BP and TUBGCP3 was confirmed by co-immunoprecipitation after co-expression of FLAG-TUBGCP3 and Myc-LGALS3BP in HEK293 cells. (d) The interactions between Myc-tagged LGALS3BP and a Flag-tagged CEP250 fragment co-expressed in HEK293 cells were confirmed by Flag-immunoprecipitation. Flag-tagged EGFP co-expressed with Myc-tagged LGALS3BP were used as negative control of the Flag-IP. (e) Endogenous MAGED2 interacts with TAP-tagged LGALS3BP. TAP-tagged EGFP was used as a negative control in the TAP experiments. (f) Examples of Coomassie stained 1-cm long SDS–PAGE gels of TAP eluates of centrosome-related bait proteins. The samples show the complexity of centrosomal protein-interacting proteins subjected to MS identification, in comparison with control samples (mock purification).

Article Snippet: Calmodulin Sepharose 4B beads (Amersham Biosciences) were washed with calmodulin-binding buffer (10 mM b-mercaptoethanol, 10 mM Hepes-KOH pH 8.0, 150 mM NaCl, 1 mM Mg(OAc)2, 1 mM imidazole, 0.1%(w/v) NP-40 and 2 mM CaCl2).

Techniques: Derivative Assay, Protein-Protein interactions, Immunoprecipitation, Binding Assay, Western Blot, Control, Expressing, Negative Control, Staining, SDS Page, Comparison