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FUJIFILM iln cells
T cell responses in DC-transferred <t>MRL/</t> lpr mice. (A) Proliferative responses of <t>ILN</t> CD4 + T cells from the recipients and control mice were analyzed. Purified CD4 + T cells were stimulated with plate-coated CD3 mAb (0–0.5 µg/ml) and CD28 mAb (10 µg/ml) for 72 hours. The proliferative response was evaluated by [ 3 H] thymidine incorporation. Values are means ± SD (n = 4, 5, and 5 respectively per group). Results are representative of three independent experiments with similar results. (B) The culture supernatants for 24 h (anti-CD3 mAb: 0.5 µg/ml; anti-CD28 mAb: 10 µg/ml) as described above were analyzed for cytokine productions including IL-2, IFN-γ, IL-4, IL-10, and IL-17 by ELISA. Values are means ± SD (n = 4, 5, and 5 respectively per group). *p
Iln Cells, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 88/100, based on 1058 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Fas-Independent T-Cell Apoptosis by Dendritic Cells Controls Autoimmune Arthritis in MRL/lpr Mice"

Article Title: Fas-Independent T-Cell Apoptosis by Dendritic Cells Controls Autoimmune Arthritis in MRL/lpr Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048798

T cell responses in DC-transferred MRL/ lpr mice. (A) Proliferative responses of ILN CD4 + T cells from the recipients and control mice were analyzed. Purified CD4 + T cells were stimulated with plate-coated CD3 mAb (0–0.5 µg/ml) and CD28 mAb (10 µg/ml) for 72 hours. The proliferative response was evaluated by [ 3 H] thymidine incorporation. Values are means ± SD (n = 4, 5, and 5 respectively per group). Results are representative of three independent experiments with similar results. (B) The culture supernatants for 24 h (anti-CD3 mAb: 0.5 µg/ml; anti-CD28 mAb: 10 µg/ml) as described above were analyzed for cytokine productions including IL-2, IFN-γ, IL-4, IL-10, and IL-17 by ELISA. Values are means ± SD (n = 4, 5, and 5 respectively per group). *p
Figure Legend Snippet: T cell responses in DC-transferred MRL/ lpr mice. (A) Proliferative responses of ILN CD4 + T cells from the recipients and control mice were analyzed. Purified CD4 + T cells were stimulated with plate-coated CD3 mAb (0–0.5 µg/ml) and CD28 mAb (10 µg/ml) for 72 hours. The proliferative response was evaluated by [ 3 H] thymidine incorporation. Values are means ± SD (n = 4, 5, and 5 respectively per group). Results are representative of three independent experiments with similar results. (B) The culture supernatants for 24 h (anti-CD3 mAb: 0.5 µg/ml; anti-CD28 mAb: 10 µg/ml) as described above were analyzed for cytokine productions including IL-2, IFN-γ, IL-4, IL-10, and IL-17 by ELISA. Values are means ± SD (n = 4, 5, and 5 respectively per group). *p

Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay

Reduced lymphoproliferation of MRL/ lpr mice following repeated transfers of DCs. (A) Spleen and ILNs from the recipient mice are shown. Photos are representative of the recipient mice (16 weeks of age) at 12 weeks after the transfer. Values are shown as means ± SD (n = 5, 7 and 7 per group respectively). (B) The total cell number in the spleen, and ILNs is shown. Scale bar: 5 mm. (C) T cell numbers in the spleen and ILNs of the recipient mice. Flow cytometry was performed using spleen and ILN cells. The number of CD4 + and CD8 + T cells is shown. (D) B cell (B220 + Thy1.2 − ) number in the spleen and ILNs of the recipient mice. (E) CD4 − CD8 − CD3 + DNT cell number in the spleen and ILNs of the recipient mice. Values are shown as means ± SD (n = 5, 7, and 7 respectively per group). *p
Figure Legend Snippet: Reduced lymphoproliferation of MRL/ lpr mice following repeated transfers of DCs. (A) Spleen and ILNs from the recipient mice are shown. Photos are representative of the recipient mice (16 weeks of age) at 12 weeks after the transfer. Values are shown as means ± SD (n = 5, 7 and 7 per group respectively). (B) The total cell number in the spleen, and ILNs is shown. Scale bar: 5 mm. (C) T cell numbers in the spleen and ILNs of the recipient mice. Flow cytometry was performed using spleen and ILN cells. The number of CD4 + and CD8 + T cells is shown. (D) B cell (B220 + Thy1.2 − ) number in the spleen and ILNs of the recipient mice. (E) CD4 − CD8 − CD3 + DNT cell number in the spleen and ILNs of the recipient mice. Values are shown as means ± SD (n = 5, 7, and 7 respectively per group). *p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

2) Product Images from "Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells"

Article Title: Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1217039110

Impaired neutrophil development from SCN-iPS cells. ( A –C) A hematopoietic colony assay was performed by using 1 × 10 4 CD34 + cells derived from three SCN-iPS cell clones (SPN0101, SPN0102, and SPN0103) and three control iPS cell clones (controls 1, 2, and 3) in the presence of a cytokine mixture. Colonies were sorted as myeloid ( A ), erythroid ( B ), and mixed-lineage (Mix) ( C ). Data are shown as mean ± SD. ( D ) Photographs of colonies ( Left ; 100×) and cells in a GM colony ( Right ; 400×; May–Grünwald–Giemsa staining). ( E ) A hematopoietic colony assay with dose escalation of G-CSF was performed by using 1 × 10 5 CD34 + cells derived from SCN-iPS and control iPS cells. Filled and open bars indicate small colonies consisting of
Figure Legend Snippet: Impaired neutrophil development from SCN-iPS cells. ( A –C) A hematopoietic colony assay was performed by using 1 × 10 4 CD34 + cells derived from three SCN-iPS cell clones (SPN0101, SPN0102, and SPN0103) and three control iPS cell clones (controls 1, 2, and 3) in the presence of a cytokine mixture. Colonies were sorted as myeloid ( A ), erythroid ( B ), and mixed-lineage (Mix) ( C ). Data are shown as mean ± SD. ( D ) Photographs of colonies ( Left ; 100×) and cells in a GM colony ( Right ; 400×; May–Grünwald–Giemsa staining). ( E ) A hematopoietic colony assay with dose escalation of G-CSF was performed by using 1 × 10 5 CD34 + cells derived from SCN-iPS and control iPS cells. Filled and open bars indicate small colonies consisting of

Techniques Used: Hematopoietic Colony Assay, Derivative Assay, Clone Assay, Staining

3) Product Images from "Fas-Independent T-Cell Apoptosis by Dendritic Cells Controls Autoimmune Arthritis in MRL/lpr Mice"

Article Title: Fas-Independent T-Cell Apoptosis by Dendritic Cells Controls Autoimmune Arthritis in MRL/lpr Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048798

T cell responses in DC-transferred MRL/ lpr mice. (A) Proliferative responses of ILN CD4 + T cells from the recipients and control mice were analyzed. Purified CD4 + T cells were stimulated with plate-coated CD3 mAb (0–0.5 µg/ml) and CD28 mAb (10 µg/ml) for 72 hours. The proliferative response was evaluated by [ 3 H] thymidine incorporation. Values are means ± SD (n = 4, 5, and 5 respectively per group). Results are representative of three independent experiments with similar results. (B) The culture supernatants for 24 h (anti-CD3 mAb: 0.5 µg/ml; anti-CD28 mAb: 10 µg/ml) as described above were analyzed for cytokine productions including IL-2, IFN-γ, IL-4, IL-10, and IL-17 by ELISA. Values are means ± SD (n = 4, 5, and 5 respectively per group). *p
Figure Legend Snippet: T cell responses in DC-transferred MRL/ lpr mice. (A) Proliferative responses of ILN CD4 + T cells from the recipients and control mice were analyzed. Purified CD4 + T cells were stimulated with plate-coated CD3 mAb (0–0.5 µg/ml) and CD28 mAb (10 µg/ml) for 72 hours. The proliferative response was evaluated by [ 3 H] thymidine incorporation. Values are means ± SD (n = 4, 5, and 5 respectively per group). Results are representative of three independent experiments with similar results. (B) The culture supernatants for 24 h (anti-CD3 mAb: 0.5 µg/ml; anti-CD28 mAb: 10 µg/ml) as described above were analyzed for cytokine productions including IL-2, IFN-γ, IL-4, IL-10, and IL-17 by ELISA. Values are means ± SD (n = 4, 5, and 5 respectively per group). *p

Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay

T-cell apoptosis via TRAIL/TRAIL-R2. (A) Real-time RT-PCR for a wide array of apoptosis-related genes was performed using mRNA samples of MRL/ lpr CD4 + T cells repeatedly stimulated with MRL/ lpr and MRL+/+ BMDCs. Gene expression of CD4 + T cells repeatedly stimulated with MRL/ lpr BMDCs was compared with those stimulated with MRL+/+ BMDCs (controls). Genes with increased and decreased expression are shown as fold of control. The experiments were repeated twice with similar results. (B) The mRNA expression of TRAF3, TRAIL-R2, caspase 8, and Bcl-2 was confirmed by quantitative real-time PCR analysis. Relative expression to β-actin level is shown as means ± SD from triplicate samples. The experiments were repeated three times with similar results. (C) Up-regulation of TRAIL expression on MRL/ lpr BMDCs by RANKL was detected by flow cytometry. Staining of DC with FITC-labeled isotype control Ab is shown as a dotted line. The experiments were repeated three times with similar results. (D) lpr CD4+ T cells were repeatedly co-cultured with activated lpr DCs in the presence of anti-TRAIL mAb. Data are shown as means ± SD of triplicate samples. *p
Figure Legend Snippet: T-cell apoptosis via TRAIL/TRAIL-R2. (A) Real-time RT-PCR for a wide array of apoptosis-related genes was performed using mRNA samples of MRL/ lpr CD4 + T cells repeatedly stimulated with MRL/ lpr and MRL+/+ BMDCs. Gene expression of CD4 + T cells repeatedly stimulated with MRL/ lpr BMDCs was compared with those stimulated with MRL+/+ BMDCs (controls). Genes with increased and decreased expression are shown as fold of control. The experiments were repeated twice with similar results. (B) The mRNA expression of TRAF3, TRAIL-R2, caspase 8, and Bcl-2 was confirmed by quantitative real-time PCR analysis. Relative expression to β-actin level is shown as means ± SD from triplicate samples. The experiments were repeated three times with similar results. (C) Up-regulation of TRAIL expression on MRL/ lpr BMDCs by RANKL was detected by flow cytometry. Staining of DC with FITC-labeled isotype control Ab is shown as a dotted line. The experiments were repeated three times with similar results. (D) lpr CD4+ T cells were repeatedly co-cultured with activated lpr DCs in the presence of anti-TRAIL mAb. Data are shown as means ± SD of triplicate samples. *p

Techniques Used: Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Labeling, Cell Culture

Fas-independent T-cell apoptosis in DC-transferred MRL/ lpr mice. (A) Experimental protocol of T cell apoptosis by repeated co-culture with DCs. Total T cells from MRL/ lpr mice (5×10 4 ) were repeatedly (three times) co-cultured with BMDCs (2.5×10 5 ) from MRL+/+ or MRL/ lpr mice for 8 hours without interval. BMDCs were stimulated with RANKL and CII for 24 hours before the co-culturing. (B) After the third co-culture, apoptosis of CD4 + and CD8 + T cells expressing annexin-V was detected by flow cytometry. Staining of T cell with FITC-labeled isotype control Ab is shown as a dotted line. Results are shown as representative of three independent experiments with similar results. (C) MFI of annexin-V on CD4 + T cells was calculated, and the data are shown as the means ± SD of triplicate samples. (D) Induction of T-cell apoptosis by repeated co-culturing with activated DCs. Purified CD4 + , CD8 + , DNT, and B220 + cells (5×10 4 ) were repeatedly co-cultured with BMDCs (5 and 25×10 4 ). Annexin-V + cells are shown as the means ± SD of triplicate samples. The experiments were repeated three times with similar results. *p
Figure Legend Snippet: Fas-independent T-cell apoptosis in DC-transferred MRL/ lpr mice. (A) Experimental protocol of T cell apoptosis by repeated co-culture with DCs. Total T cells from MRL/ lpr mice (5×10 4 ) were repeatedly (three times) co-cultured with BMDCs (2.5×10 5 ) from MRL+/+ or MRL/ lpr mice for 8 hours without interval. BMDCs were stimulated with RANKL and CII for 24 hours before the co-culturing. (B) After the third co-culture, apoptosis of CD4 + and CD8 + T cells expressing annexin-V was detected by flow cytometry. Staining of T cell with FITC-labeled isotype control Ab is shown as a dotted line. Results are shown as representative of three independent experiments with similar results. (C) MFI of annexin-V on CD4 + T cells was calculated, and the data are shown as the means ± SD of triplicate samples. (D) Induction of T-cell apoptosis by repeated co-culturing with activated DCs. Purified CD4 + , CD8 + , DNT, and B220 + cells (5×10 4 ) were repeatedly co-cultured with BMDCs (5 and 25×10 4 ). Annexin-V + cells are shown as the means ± SD of triplicate samples. The experiments were repeated three times with similar results. *p

Techniques Used: Mouse Assay, Co-Culture Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Staining, Labeling, Purification

Reduced lymphoproliferation of MRL/ lpr mice following repeated transfers of DCs. (A) Spleen and ILNs from the recipient mice are shown. Photos are representative of the recipient mice (16 weeks of age) at 12 weeks after the transfer. Values are shown as means ± SD (n = 5, 7 and 7 per group respectively). (B) The total cell number in the spleen, and ILNs is shown. Scale bar: 5 mm. (C) T cell numbers in the spleen and ILNs of the recipient mice. Flow cytometry was performed using spleen and ILN cells. The number of CD4 + and CD8 + T cells is shown. (D) B cell (B220 + Thy1.2 − ) number in the spleen and ILNs of the recipient mice. (E) CD4 − CD8 − CD3 + DNT cell number in the spleen and ILNs of the recipient mice. Values are shown as means ± SD (n = 5, 7, and 7 respectively per group). *p
Figure Legend Snippet: Reduced lymphoproliferation of MRL/ lpr mice following repeated transfers of DCs. (A) Spleen and ILNs from the recipient mice are shown. Photos are representative of the recipient mice (16 weeks of age) at 12 weeks after the transfer. Values are shown as means ± SD (n = 5, 7 and 7 per group respectively). (B) The total cell number in the spleen, and ILNs is shown. Scale bar: 5 mm. (C) T cell numbers in the spleen and ILNs of the recipient mice. Flow cytometry was performed using spleen and ILN cells. The number of CD4 + and CD8 + T cells is shown. (D) B cell (B220 + Thy1.2 − ) number in the spleen and ILNs of the recipient mice. (E) CD4 − CD8 − CD3 + DNT cell number in the spleen and ILNs of the recipient mice. Values are shown as means ± SD (n = 5, 7, and 7 respectively per group). *p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

Regulation of Fas-independent T cell apoptosis by TRAIL siRNA-treated DCs. (A) BMDCs from MRL/ lpr mice were treated with TRAIL gene-specific siRNA or control siRNA for 24 hours, and then stimulated with RANKL and CII for 48 hours. Purified CD4 + T cells of LNs from MRL/ lpr mice were repeatedly (three times) co-cultured with the activated DCs for 8 hours by transfer into each new well. (B) Expression of TRAIL on activated BMDCs treated with TRAIL siRNA or control siRNA was analyzed by flow cytometry. Results are representative of two independent experiments with similar results. (C) Apoptosis of CD4 + T cells cocultured with siRNA-treated DCs was analyzed by flow cytometry with Annexin-V and PI. Results are representative of two independent experiments. (D) Apoptotic cells (%) are shown as the mean ± SD from triplicate samples. The experiments were repeated three times with similar results. (E) In vitro TRAIL siRNA-treated and control DCs were injected three times into MRL/ lpr mice (4 weeks of age). After 12 weeks after the transfers, RF level of sera from the recipient mice (16 weeks of age) was detected by ELISA. Values are means ± SD. (n = 5). The experiments were repeated twice with similar results. (F) Histology of joint from recipient mice. Histological photos with HE staining are shown as representative of five mice in each group at 12 weeks after transfers. Arrow; bone erosion or synovial proliferation, star; mononuclear inflammatory infiltrate, fibrosis, or panus. Scale bar: 100 µm. Histological score is shown as means ± SD. (n = 5) *p
Figure Legend Snippet: Regulation of Fas-independent T cell apoptosis by TRAIL siRNA-treated DCs. (A) BMDCs from MRL/ lpr mice were treated with TRAIL gene-specific siRNA or control siRNA for 24 hours, and then stimulated with RANKL and CII for 48 hours. Purified CD4 + T cells of LNs from MRL/ lpr mice were repeatedly (three times) co-cultured with the activated DCs for 8 hours by transfer into each new well. (B) Expression of TRAIL on activated BMDCs treated with TRAIL siRNA or control siRNA was analyzed by flow cytometry. Results are representative of two independent experiments with similar results. (C) Apoptosis of CD4 + T cells cocultured with siRNA-treated DCs was analyzed by flow cytometry with Annexin-V and PI. Results are representative of two independent experiments. (D) Apoptotic cells (%) are shown as the mean ± SD from triplicate samples. The experiments were repeated three times with similar results. (E) In vitro TRAIL siRNA-treated and control DCs were injected three times into MRL/ lpr mice (4 weeks of age). After 12 weeks after the transfers, RF level of sera from the recipient mice (16 weeks of age) was detected by ELISA. Values are means ± SD. (n = 5). The experiments were repeated twice with similar results. (F) Histology of joint from recipient mice. Histological photos with HE staining are shown as representative of five mice in each group at 12 weeks after transfers. Arrow; bone erosion or synovial proliferation, star; mononuclear inflammatory infiltrate, fibrosis, or panus. Scale bar: 100 µm. Histological score is shown as means ± SD. (n = 5) *p

Techniques Used: Mouse Assay, Purification, Cell Culture, Expressing, Flow Cytometry, Cytometry, In Vitro, Injection, Enzyme-linked Immunosorbent Assay, Staining

Therapeutic effect of repeated transfers of DCs on autoimmune arthritis. (A) Experimental protocol is shown. BMDCs from female MRL+/+ and MRL/ lpr mice were stimulated with RANKL and CII, and then female MRL/ lpr mice received a total of 3 injections of the BMDCs every other day distributed over 6 day period. At 16 weeks after transfer (20 weeks of age), the recipient MRL/ lpr mice were analyzed. (B) Histology of joint from recipient mice. Histological photos with HE staining are shown as representative of the recipient mice at 16 weeks after transfers. Arrow; bone erosion or synovial proliferation, star; mononuclear inflammatory infiltrate, fibrosis, or panus. Scale bar: 100 µm (n = 7, 10 and 12 per group respectively). (C) The histological score of the recipient mice was evaluated at 16 weeks after repeated transfers. Data are shown as means ± SD. (n = 7, 10 and 12 per group respectively). (D) Rheumatoid factor (RF) (IgM and IgG) antibody was measured by ELISA. Values are shown as means ± SD (n = 7, 10 and 12 per group respectively). OD = optical density. (E) RA lesions of control, a single DC transferred (1× DC), and multiple DC transferred (3× DC) MRL/ lpr mice were compared. Histological photos with HE staining are shown as representative of the recipient mice at 12 weeks after transfers. Scale bar: 100 µm (n = 5 per group respectively). (F) The histological score of the recipient mice was evaluated at 12 weeks after repeated transfers. Data are shown as means ± SD (n = 5 per group respectively). *p
Figure Legend Snippet: Therapeutic effect of repeated transfers of DCs on autoimmune arthritis. (A) Experimental protocol is shown. BMDCs from female MRL+/+ and MRL/ lpr mice were stimulated with RANKL and CII, and then female MRL/ lpr mice received a total of 3 injections of the BMDCs every other day distributed over 6 day period. At 16 weeks after transfer (20 weeks of age), the recipient MRL/ lpr mice were analyzed. (B) Histology of joint from recipient mice. Histological photos with HE staining are shown as representative of the recipient mice at 16 weeks after transfers. Arrow; bone erosion or synovial proliferation, star; mononuclear inflammatory infiltrate, fibrosis, or panus. Scale bar: 100 µm (n = 7, 10 and 12 per group respectively). (C) The histological score of the recipient mice was evaluated at 16 weeks after repeated transfers. Data are shown as means ± SD. (n = 7, 10 and 12 per group respectively). (D) Rheumatoid factor (RF) (IgM and IgG) antibody was measured by ELISA. Values are shown as means ± SD (n = 7, 10 and 12 per group respectively). OD = optical density. (E) RA lesions of control, a single DC transferred (1× DC), and multiple DC transferred (3× DC) MRL/ lpr mice were compared. Histological photos with HE staining are shown as representative of the recipient mice at 12 weeks after transfers. Scale bar: 100 µm (n = 5 per group respectively). (F) The histological score of the recipient mice was evaluated at 12 weeks after repeated transfers. Data are shown as means ± SD (n = 5 per group respectively). *p

Techniques Used: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay

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    FUJIFILM 2 mercaptoethanol
    Autoubiquitination activity of mE2-C. (A) Recombinant wild-type (wt) or mutant (C114S) mE2-C (20 kDa) was incubated with 125 I-ubiquitin in the absence or presence of E1 (116 kDa). The reaction mixtures then were boiled in SDS sample buffer, without (upper panel) or with (lower panel) <t>2-mercaptoethanol</t> (2ME), and were subjected to SDS-PAGE and autoradiography. The positions of free ubiquitin (Ub), E1 conjugated with ubiquitin (E1-Ub), mE2-C conjugated with ubiquitin (mE2-C-Ub), and GST-mE2-C conjugated with ubiquitin (GST-mE2-C-Ub) are indicated on the right, and those of molecular size standards (in kilodaltons) are shown on the left. (B) Cell lysates prepared from mouse NIH 3T3 and human HeLa cells were subjected to immunoblot analysis with anti-mE2-C. The positions of the immunoreactive mE2-C and hE2-C proteins are indicated. (C) NIH 3T3 cells were transfected with either an expression plasmid encoding Myc-tagged mE2-C or the empty vector (pcDNA3). Cell lysates were subsequently prepared and subjected to immunoblot analysis (IB) with anti-mE2-C, anti-Myc, or antiubiquitin. The positions of mE2-C, Myc-mE2-C, and monoubiquitinated Myc-mE2-C (Myc-mE2-C-Ub) are shown. The asterisk indicates a nonspecific band. Because the exposure time was shorter for lane 3 than for lane 1, the endogenous mE2-C is not visible in lane 3.
    2 Mercaptoethanol, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 94/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/2 mercaptoethanol/product/FUJIFILM
    Average 94 stars, based on 105 article reviews
    Price from $9.99 to $1999.99
    2 mercaptoethanol - by Bioz Stars, 2020-07
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    93
    FUJIFILM beta mercaptoethanol
    Autoubiquitination activity of mE2-C. (A) Recombinant wild-type (wt) or mutant (C114S) mE2-C (20 kDa) was incubated with 125 I-ubiquitin in the absence or presence of E1 (116 kDa). The reaction mixtures then were boiled in SDS sample buffer, without (upper panel) or with (lower panel) <t>2-mercaptoethanol</t> (2ME), and were subjected to SDS-PAGE and autoradiography. The positions of free ubiquitin (Ub), E1 conjugated with ubiquitin (E1-Ub), mE2-C conjugated with ubiquitin (mE2-C-Ub), and GST-mE2-C conjugated with ubiquitin (GST-mE2-C-Ub) are indicated on the right, and those of molecular size standards (in kilodaltons) are shown on the left. (B) Cell lysates prepared from mouse NIH 3T3 and human HeLa cells were subjected to immunoblot analysis with anti-mE2-C. The positions of the immunoreactive mE2-C and hE2-C proteins are indicated. (C) NIH 3T3 cells were transfected with either an expression plasmid encoding Myc-tagged mE2-C or the empty vector (pcDNA3). Cell lysates were subsequently prepared and subjected to immunoblot analysis (IB) with anti-mE2-C, anti-Myc, or antiubiquitin. The positions of mE2-C, Myc-mE2-C, and monoubiquitinated Myc-mE2-C (Myc-mE2-C-Ub) are shown. The asterisk indicates a nonspecific band. Because the exposure time was shorter for lane 3 than for lane 1, the endogenous mE2-C is not visible in lane 3.
    Beta Mercaptoethanol, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/beta mercaptoethanol/product/FUJIFILM
    Average 93 stars, based on 2 article reviews
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    Autoubiquitination activity of mE2-C. (A) Recombinant wild-type (wt) or mutant (C114S) mE2-C (20 kDa) was incubated with 125 I-ubiquitin in the absence or presence of E1 (116 kDa). The reaction mixtures then were boiled in SDS sample buffer, without (upper panel) or with (lower panel) 2-mercaptoethanol (2ME), and were subjected to SDS-PAGE and autoradiography. The positions of free ubiquitin (Ub), E1 conjugated with ubiquitin (E1-Ub), mE2-C conjugated with ubiquitin (mE2-C-Ub), and GST-mE2-C conjugated with ubiquitin (GST-mE2-C-Ub) are indicated on the right, and those of molecular size standards (in kilodaltons) are shown on the left. (B) Cell lysates prepared from mouse NIH 3T3 and human HeLa cells were subjected to immunoblot analysis with anti-mE2-C. The positions of the immunoreactive mE2-C and hE2-C proteins are indicated. (C) NIH 3T3 cells were transfected with either an expression plasmid encoding Myc-tagged mE2-C or the empty vector (pcDNA3). Cell lysates were subsequently prepared and subjected to immunoblot analysis (IB) with anti-mE2-C, anti-Myc, or antiubiquitin. The positions of mE2-C, Myc-mE2-C, and monoubiquitinated Myc-mE2-C (Myc-mE2-C-Ub) are shown. The asterisk indicates a nonspecific band. Because the exposure time was shorter for lane 3 than for lane 1, the endogenous mE2-C is not visible in lane 3.

    Journal: Molecular Biology of the Cell

    Article Title: Cell Cycle-Dependent Expression of Mammalian E2-C Regulated by the Anaphase-Promoting Complex/Cyclosome

    doi:

    Figure Lengend Snippet: Autoubiquitination activity of mE2-C. (A) Recombinant wild-type (wt) or mutant (C114S) mE2-C (20 kDa) was incubated with 125 I-ubiquitin in the absence or presence of E1 (116 kDa). The reaction mixtures then were boiled in SDS sample buffer, without (upper panel) or with (lower panel) 2-mercaptoethanol (2ME), and were subjected to SDS-PAGE and autoradiography. The positions of free ubiquitin (Ub), E1 conjugated with ubiquitin (E1-Ub), mE2-C conjugated with ubiquitin (mE2-C-Ub), and GST-mE2-C conjugated with ubiquitin (GST-mE2-C-Ub) are indicated on the right, and those of molecular size standards (in kilodaltons) are shown on the left. (B) Cell lysates prepared from mouse NIH 3T3 and human HeLa cells were subjected to immunoblot analysis with anti-mE2-C. The positions of the immunoreactive mE2-C and hE2-C proteins are indicated. (C) NIH 3T3 cells were transfected with either an expression plasmid encoding Myc-tagged mE2-C or the empty vector (pcDNA3). Cell lysates were subsequently prepared and subjected to immunoblot analysis (IB) with anti-mE2-C, anti-Myc, or antiubiquitin. The positions of mE2-C, Myc-mE2-C, and monoubiquitinated Myc-mE2-C (Myc-mE2-C-Ub) are shown. The asterisk indicates a nonspecific band. Because the exposure time was shorter for lane 3 than for lane 1, the endogenous mE2-C is not visible in lane 3.

    Article Snippet: After incubation for 15 min at 22°C, the reaction mixtures were heated at 95°C for 5 min in SDS sample buffer with or without 5% (vol/vol) 2-mercaptoethanol and then were subjected to SDS-PAGE on a 12% gel, autoradiography, and image analysis (BAS-2000, Fuji Film, Tokyo, Japan).

    Techniques: Activity Assay, Recombinant, Mutagenesis, Incubation, SDS Page, Autoradiography, Transfection, Expressing, Plasmid Preparation