Journal: Human molecular genetics

Article Title: FcgammaRIIB Ile232Thr transmembrane polymorphism associated with human systemic lupus erythematosus decreases affinity to lipid rafts and attenuates inhibitory effects on B cell receptor signaling.

doi: 10.1093/hmg/ddi320

Figure Lengend Snippet: Figure 3. BCR-mediated calcium mobilization was less inhibited by c-232Thr than by c-232Ile. (A) Time-dependent changes in [Ca2þ]i in c-232Ile- and c-232Thr-expressing cells stimulated with anti-IgM F(ab0)2 (BCR-alone, upper panel) or with whole IgG (coligation, lower panel). The pair of traces at each panel represents the average of four independent experiments of the four different clones pairs (n ¼ 16). (B) [Ca2þ]i values at the peak (120 s after the stimu- lation) and plateau (400 s) phases in six experiments of parent cells (n ¼ 6), and four experiments using the four different clone pairs expressing c-232Ile (n ¼ 16) or c-232Thr (n ¼ 16). To avoid complexity, statistical comparison between the data from the c-232Ile- and c-232Thr-expressing cells is presented (mean + SD, P , 0.01, P , 0.05).

Article Snippet: Antibodies used for probing or ligation of BCR and FcgRIIB Anti-human IgM rabbit whole IgG and anti-human IgM rabbit IgG F(ab0)2 were obtained from Jackson Immunoresearch.

Techniques: Expressing, Clone Assay, Comparison

Journal: Human molecular genetics

Article Title: FcgammaRIIB Ile232Thr transmembrane polymorphism associated with human systemic lupus erythematosus decreases affinity to lipid rafts and attenuates inhibitory effects on B cell receptor signaling.

doi: 10.1093/hmg/ddi320

Figure Lengend Snippet: Figure 4. Ile232Thr substitution impairs FcgRIIB tyrosine phosphorylation and SHIP recruitment, and ability of FcgRIIB to inhibit BCR-mediated biochemical signaling. (A) c-232Ile and c-232Thr tyrosine phosphorylation, and SHIP recruitment to FcgRIIB variants. Cells were stimulated with anti-IgM F(ab0)2 (BCR alone) or with whole IgG (coligation) in the presence or absence of the pretreatment with blocking Abs (2.4G2 block) for 1 min at 378C. c-232Ile and c-232Thr were immunoprecipitated with 2.4G2, and the immunoprecipitates were subjected to immunoblotting (IB) for the analysis of FcgRIIB tyrosine phosphorylation (p-Y) and for that of SHIP co-precipitation (SHIP). After stripping, the blotted membrane was reprobed with anti-FcgRIIB polyclonal Abs for loading control. (B) Tyrosine phosphorylation of CD19 in c-232Ile- and c-232Thr-expressing cells. Cells were stimulated and processed as described in (A) and CD19 was immu- noprecipitated. The immunoprecipitates were divided into two halves, one was subjected to anti-phosphotyrosine immunoblotting (IB) and the other to anti-CD19 immunoblotting for loading control. (C) Time-dependent changes in PIP3 accumulation in c-232Ile and c-232Thr clones after BCR-FcgRIIB coligation. Phospho- lipids extracted from 32P-labeled cells were analyzed by thin-layer chromatography followed by BAS2000 (Fuji Photo Film Co., Ltd) imaging. Changes in PIP3/ PIP2 ratio (normalized by the basal value (time 0) in c-232Ile cells) in three different clone pairs are shown (mean + SD, n ¼ 3, P , 0.05). (D) Time-dependent changes in the phosphorylation of Akt Ser473 and PLCg2 Tyr1217. c-232Ile and c-232Thr clones solubilized with n-octyl-b-D-glucoside were subjected to the immunoblotting with phosphorylation-site-specific Abs. Membranes were reprobed with Abs against cognate molecules for loading control. Graphs show changes in the density of phosphorylation signals in two independent experiments using three different clone pairs. Signal density at each time point is normalized by the total signal density in the respective c-232Ile and c-232Thr pairs (mean + SD, n ¼ 6, P , 0.01, P , 0.05). (E) PLCg2 Tyr1217 phosphorylation at lipid rafts. c-232Ile and c-232Thr clones stimulated by BCR-FcgRIIB coligation were subjected to sucrose density gradient centrifugation assay and non-raft fractions (nr) and raft fractions (r) were subjected to immunoblotting with PLCg2 phosphorylation-site-specific Abs. Cbp and PLCg2 stainings were performed for the verification of raft fractions and for loading control, respectively. This blot is representative of two experiments using different clone pairs.

Article Snippet: Antibodies used for probing or ligation of BCR and FcgRIIB Anti-human IgM rabbit whole IgG and anti-human IgM rabbit IgG F(ab0)2 were obtained from Jackson Immunoresearch.

Techniques: Phospho-proteomics, Blocking Assay, Immunoprecipitation, Western Blot, Stripping Membranes, Membrane, Control, Expressing, Clone Assay, Labeling, Thin Layer Chromatography, Imaging, Gradient Centrifugation

Journal: Journal of Endocrinology

Article Title: Calcineurin expression and activity is regulated by the intracellular redox status and under hypertension in human neutrophils

doi: 10.1530/joe-12-0106

Figure Lengend Snippet: Figure 6 Effect of CN on negative modulation by Ang II of HO1 and Nrf2 nuclear translocation in human neutrophils. Neutrophils from healthy donors were grown at 37 8C in RPMI medium in the absence or presence of 0.3 g/ml CsA for 1 h (A) or 1 mg/ml CsA for 30 min (B). Then, 100 nM Ang II was added for 1.5 h (A) or 30 min (B), and thereafter, the cells were stimulated or not with 10 mM 15dPGJ2 for a further 5 h (A) or 30 min (B). The cells were then lysed and total proteins (A) or nuclear extracts (B) were obtained. Levels of HO1, GAPDH (A), and NRF2 (B) proteins were measured by immuno- blotting analysis. The bands shown are representative of a set of three independent experiments. Plotted values (meanGS.E.M.) represent total HO1 protein expression corrected for differences in GAPDH levels (A) or Nrf2 nuclear levels (B). Statistical significance: *P!0.01, for 15dPGJ2-stimulated vs unstimulated; **P!0.01, for 15dPGJ2-stimulated, Ang II-treated vs Ang II-untreated; #P!0.01, for 15dPGJ2-stimulated, Ang II- and CsA-treated vs CsA-untreated.

Article Snippet: Antibodies to HO1 (sc-10789) and Nrf2 (sc-722) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pressure (mmHg) Diastolic blood pressure (mmHg) 75.5G4.5 101.4G7.9 %0.001 Body mass index (kg/m2) 24.2G3.4 28.9G2.2 %0.05 Isolation and culture of human neutrophils Human peripheral blood neutrophils were isolated from fresh heparinized blood of human donors by dextran sedimentation Journal of Endocrinology (2012) 214, 399–408 followed by Ficoll-Paque gradient centrifugation, and hypotonic lysis of residual erythrocytes (Carballo et al. 1999).

Techniques: Translocation Assay, Expressing

Journal: Journal of Endocrinology

Article Title: Calcineurin expression and activity is regulated by the intracellular redox status and under hypertension in human neutrophils

doi: 10.1530/joe-12-0106

Figure Lengend Snippet: Figure 7 HO1 expression levels in neutrophils of hypertensive patients and effect of CsA on the expression of HO1 in these patients. Neutrophils from healthy subjects or hypertensive patients were cultured at 37 8C in RPMI medium in the absence or presence of CsA (0.3 g/ml) for 1 h and then stimulated or not with 10 mM 15dPGJ2 for further 15 h. The cells were then lysed, total proteins were obtained, and HO1 and GAPDH levels were analyzed by immunoblotting. The bands shown are representative of a set of three independent experiments. Plotted values (mean GS.E.M.) represent HO1 protein expression levels corrected for differences in GAPDH levels. Statistical significance: *P!0.001, for 15dPGJ2-stimulated in hypertensive vs 15dPGJ2-stimulated in healthy; **P!0.001, for 15dPGJ2-stimulated, CsA-treated vs 15dPGJ2-stimulated, and CsA-untreated in hypertensive.

Article Snippet: Antibodies to HO1 (sc-10789) and Nrf2 (sc-722) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pressure (mmHg) Diastolic blood pressure (mmHg) 75.5G4.5 101.4G7.9 %0.001 Body mass index (kg/m2) 24.2G3.4 28.9G2.2 %0.05 Isolation and culture of human neutrophils Human peripheral blood neutrophils were isolated from fresh heparinized blood of human donors by dextran sedimentation Journal of Endocrinology (2012) 214, 399–408 followed by Ficoll-Paque gradient centrifugation, and hypotonic lysis of residual erythrocytes (Carballo et al. 1999).

Techniques: Expressing, Cell Culture, Western Blot

Journal: Cells

Article Title: Astro-Versus Microglia-Enriched Transcriptomes from Aged Atxn2 -CAG100-Knockin Mice Suggest Underlying Pathology of RNA Processing at Ribosomes, and Possibly at U-Bodies

doi: 10.3390/cells15080699

Figure Lengend Snippet: Schematic workflow for adult brain dissociation and sequential isolation of cell types (microglia, astrocytes, and neurons). Tissue is harvested rapidly, placed in ice-cold DPBS with Ca 2+ and Mg 2+ , and cut into small pieces, then transferred to C-tubes and dissociated by a combination of enzymatic buffer solution containing papain with gentle mechanical dissociation, using a gentleMACS Octo Dissociator with heaters. Gradient centrifugation forms a compact ring containing debris (dead cells and myelin). After debris removal, red blood cells are lysed, and the dissociated cells are magnetically labeled with anti-CD11b MicroBeads against microglia. The labeled cells are passed through LS columns twice and placed on a magnetic stand. CD11b-positively selected cells remain in the column and are eluted with a plunger into a fresh tube. The negative flow-through is processed to isolate astrocytes labeled with anti-ACSA2 MicroBeads, and the process is repeated. For neuron isolation, the negative flow-through from astrocyte extraction is labeled with a non-neuronal biotin cocktail and anti-biotin MicroBeads, and the subsequent negative flow-through contains the neuron population. The arrows show the workflow handling of the cell suspensions. Image created in BioRender, Reddy, A. (2026) https://BioRender.com/mj9rqid , last accessed on 2 March 2026 and adapted from .

Article Snippet: DPBS was replaced with 1950 μL of enzyme mix 1, as mentioned in the Miltenyi adult brain dissociation kit (Miltenyi #130-107-677).

Techniques: Isolation, Gentle, Gradient Centrifugation, Labeling, Extraction