nfat2  (Proteintech)

Journal: PLOS One

Article Title: Generation of osteoclast-like cells from human peripheral blood mononuclear cells using NFATc1 modified RNA

doi: 10.1371/journal.pone.0342642

Figure Lengend Snippet: (A) Expression of NFATc1 modRNA in human macrophages at 24 hours posttransfection. Data are mean ± SEM (n = 3). * p < 0.05 tested by ANOVA with Tukey’s multiple comparison. (B) Numbers of TRAP Bright cells following treatment with Lipofectamine (control) or transfection with 50 ng/mL of NFATc1 modRNA (n = 3). Data are mean ± SEM (n = 3). # p < 0.05 tested by t-test (C, D) Representative images of the formalin-fixed, TRAP Bright cells described in (B). Scale bars = 100 µm.

Article Snippet: The membranes were blocked with 5% skimmed milk for 1 hour and subsequently incubated with primary antibodies, including NFATc1 (Cell Signaling, #8032) and β-actin (Cell Signaling, #4970) at 4 °C overnight.

Techniques: Expressing, Comparison, Control, Transfection

Journal: PLOS One

Article Title: Generation of osteoclast-like cells from human peripheral blood mononuclear cells using NFATc1 modified RNA

doi: 10.1371/journal.pone.0342642

Figure Lengend Snippet: Representative fluorescence microscopy images of F-actin formation in human macrophages on day 1 (A) , 2 (B) and 3 (C) posttransfection of NFATc1 modRNA. NFATc1 modRNA at 50 ng/mL was transfected in human macrophages and stained with iFluoro488-conjugated phalloidin (green) in human macrophages (n = 3). Scale bars = 50 µm.

Article Snippet: The membranes were blocked with 5% skimmed milk for 1 hour and subsequently incubated with primary antibodies, including NFATc1 (Cell Signaling, #8032) and β-actin (Cell Signaling, #4970) at 4 °C overnight.

Techniques: Fluorescence, Microscopy, Transfection, Staining

Journal: PLOS One

Article Title: Generation of osteoclast-like cells from human peripheral blood mononuclear cells using NFATc1 modified RNA

doi: 10.1371/journal.pone.0342642

Figure Lengend Snippet: Representative confocal microscopy images showing upregulated F-actin formation and cathepsin K expression in human macrophages at 3 days posttransfection of 50 ng/mL NFATc1 modRNA.

Article Snippet: The membranes were blocked with 5% skimmed milk for 1 hour and subsequently incubated with primary antibodies, including NFATc1 (Cell Signaling, #8032) and β-actin (Cell Signaling, #4970) at 4 °C overnight.

Techniques: Confocal Microscopy, Expressing

Journal: PLOS One

Article Title: Generation of osteoclast-like cells from human peripheral blood mononuclear cells using NFATc1 modified RNA

doi: 10.1371/journal.pone.0342642

Figure Lengend Snippet: (A) Representative images of resorptive pits (empty spaces around multinucleated cells; arrows) in human macrophages transfected with Lipofectamine (control) or 50 ng/mL NFATc1 modRNA and cultured on a FACS calcium phosphate-coated plate for 3 days. Scale bars = 50 µm. (B) Free calcium phosphate levels in the culture medium of (A); * p < 0.05, analyzed by t-test (n = 3).

Article Snippet: The membranes were blocked with 5% skimmed milk for 1 hour and subsequently incubated with primary antibodies, including NFATc1 (Cell Signaling, #8032) and β-actin (Cell Signaling, #4970) at 4 °C overnight.

Techniques: Transfection, Control, Cell Culture

Journal: PLOS One

Article Title: Generation of osteoclast-like cells from human peripheral blood mononuclear cells using NFATc1 modified RNA

doi: 10.1371/journal.pone.0342642

Figure Lengend Snippet: SEM micrographs of Lipofectamine (control) or 50 ng/mL NFATc1 modRNA-induced osteoclasts at day 3 posttransfection (n = 3). Red arrow indicates osteoclast with microvilli projections. Scale bars = 50 µm.

Article Snippet: The membranes were blocked with 5% skimmed milk for 1 hour and subsequently incubated with primary antibodies, including NFATc1 (Cell Signaling, #8032) and β-actin (Cell Signaling, #4970) at 4 °C overnight.

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Mitochondrial calcium shapes B cell signaling and mitochondrial function

doi: 10.3389/fimmu.2025.1710128

Figure Lengend Snippet: The loss of MCU alters cell signaling and oxygen consumption. (A–G) Mcu fl/fl x mb1 CreERT2 (MCU -/- ) and control (Ctrl) mice were injected with tamoxifen for three consecutive days to induce B cell-specific Mcu deletion. (A) Representative immunoblot analysis of MCU protein levels in MCU -/- and Ctrl mouse B cells stimulated with anti-IgM for 2 days. Representative of 3 independent experiments. (B) Mitochondrial Ca 2+ levels in MCU -/- and Ctrl mouse B cells stimulated with anti-mouse IgM (10µg/ml) for 1 (left) and 2 days (right) were determined by Rhod-2 AM staining. Pooled data from 6 and 5 independent experiments, respectively. (d1: n=9 for MCU -/- and 10 for Ctrl, d2: n=8 for MCU -/- and Ctrl mice). (C) Measurement of mouse B cell proliferation by eFluor 670 dilution. MCU -/- and Ctrl mouse B cells were stimulated with anti- IgM and tracked for 3 days. Representative of 3 independent experiments. (n=6 MCU -/- and 5 Ctrl mice). (D) Cell survival analysis of mouse B cells shown in (C) . Forward scatter (FSC) and side scatter (SSC) properties were used to determine the percentage of living cells. Pooled data from 3 experiments. (n=6 MCU -/- and 5 Ctrl mice). (E) Cytosolic Ca 2+ was assessed in MCU -/- and Ctrl B cells using indo-1 AM. Shown are basal and peak Ca 2+ levels after anti-IgM stimulation as well as the difference between peak and basal levels (delta). Pooled data from 5 independent experiments. (n=7 for MCU -/- and for Ctrl). (F) Representative immunoblot analysis of NFAT2 protein levels in MCU -/- mouse B cells compared to Ctrl B cells. Cells were stimulated with anti-mouse IgM (10µg/ml) for 48 h. Blots were probed for NFAT2 and actin. Representative of 3 independent experiments. (G) Quantification of (F) was performed by normalization of NFAT2 levels to actin levels. Then, fold change in NFAT2 levels of MCU -/- mouse B cells relative to Ctrl B cells was calculated. Pooled data from 3 independent experiments. (n=5 Ctrl and 7 MCU -/- mice). (H–N) Mcu fl/fl and Ctrl B cells were treated with TAT-CRE and stimulated with anti-IgM. (H) Representative immunoblot analysis of MCU protein levels in MCU -/- and Ctrl mouse B cells stimulated with anti-IgM for 48h. Blots were probed with MCU and actin antibodies. Representative of 3 independent experiments. (I) Mitochondrial Ca 2+ levels in MCU -/- and Ctrl mouse B cells stimulated as in (H) was determined with Rhod-2 AM staining. Pooled data from 5 independent experiments. (n=6 MCU -/- and 7 Ctrl mice). (J) MCU -/- and Ctrl B cells were stimulated with anti-IgM. Proliferation was assessed by eFluor 670 dilution. Representative of 3 independent experiments. (K) Representative immunoblot analysis of NFAT2 protein expression in MCU -/- and Ctrl mouse B cells shown in (H) . Blots were probed for NFAT2 and actin. Representative of 3 independent experiments. (L) Quantification of (K) was performed by normalization of NFAT2 levels to actin levels. Then, fold change of NFAT2 levels of MCU-deficient mouse B cells relative to Ctrl B cells was calculated. Pooled data from 3 independent repeats. (n=4 for MCU -/- and Ctrl mice). (M) Representative oxygen consumption measurement of MCU -/- and Ctrl mouse B cells as in (H) . Oxygen consumption rates (OCR) were measured by Seahorse flux technology after sequential injection of oligomycin (1µM), FCCP (1µM) and rotenone + antimycin A (1µM). Representative of 3 independent experiments. (N) Summary graphs showing calculated basal and maximal respiration in anti-IgM stimulated MCU-deficient and Ctrl mouse B cells shown in (M) . Pooled data from 3 independent experiments. (n=3 for MCU -/- and Ctrl). Data are presented as mean. Paired (B, I, N) and unpaired (G, L) Student’s t tests were used to compare groups. *p < 0.05; ns, not significant.

Article Snippet: Membranes were probed with antibodies against MCU, MCUR1, NFAT2, total and cleaved caspase 3, pPLCγ2(Y1217), pBtk(Y223), pS6(S235/236), pAkt(S473), PLCγ2, Btk, Akt (Cell Signaling Technology), and β-actin (Proteintech) overnight at 4°C.

Techniques: Control, Injection, Western Blot, Staining, Expressing