bti pht315  (ATCC)

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Oligonucleotides used for Real-Time Quantitative PCR.

Article Snippet: Immunoprecipitated proteins were subjected to Western analysis as above, using primary antibodies specific for HDAC1 (C19) or Nab2 (IC4) (both from Santa Cruz).

Techniques:

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Confluent cultures of human foreskin fibroblasts were incubated with TGF-β1 (10 ng/ml) for indicated periods. A, D Total RNA was isolated and analysed by real-time quantitative PCR. Results, normalized with actin, are the means±S.D. of triplicate determinations from a representative experiment. B. Whole cell lysates were subjected to Western analysis. Representative immunoblots. C. Fibroblasts were fixed and stained with anti-Nab2 antibodies, or DAPI to detect nuclei, and viewed by immunofluorescence microscopy (original magnification ×100).

Article Snippet: Immunoprecipitated proteins were subjected to Western analysis as above, using primary antibodies specific for HDAC1 (C19) or Nab2 (IC4) (both from Santa Cruz).

Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Staining, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Cultures of fibroblasts were cotransfected with Nab2 expression vectors or empty vector and harvested following incubation with TGF-β1 for 24 or 48 h. A. Whole cell lysates were examined by Western analysis. Representative immunoblots. B. Fibroblasts were fixed, incubated with antibodies to Type I collagen, and examined by immunofluorescence microscopy (original magnification ×400). Nuclei were identified by DAPI (blue). C. Total RNA was isolated and examined by real-time qPCR. Results, normalized with actin, are the means±S.D. of triplicate determinations from a representative experiment. D. Fibroblasts were cotransfected with 772COL1A2-CAT. Cell lysates were assayed for their CAT activities (upper panels) or examined by Western analysis (lower panels). Results of CAT assays, normalized with Renilla luciferase, are expressed as means±S.D. of triplicate determinations from a representative experiment. Open boxes, untreated fibroblasts; closed boxes, TGF-β-treated fibroblasts. *p<0.005. E. After 48 h fibroblasts were fixed, incubated with antibodies to α-SMA (green) and examined by immunofluorescence microscopy. Nuclei were identified by DAPI (blue). Representative images (original magnification ×100).

Article Snippet: Immunoprecipitated proteins were subjected to Western analysis as above, using primary antibodies specific for HDAC1 (C19) or Nab2 (IC4) (both from Santa Cruz).

Techniques: Expressing, Plasmid Preparation, Incubation, Western Blot, Immunofluorescence, Microscopy, Isolation, Luciferase

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Wildtype and Nab2 −/− mouse embryonic fibroblasts (MEFs) cultured in parallel were incubated with or without TGF-β1 for 24 h. A. Whole cell lysates and culture supernatants were subjected to Western analysis. Representative immunoblots. B. Total RNA was subjected to real-time qPCR analysis. Results, expressed relative to 18 s, are the means±S.D. of triplicate determinations from a representative experiment. Open boxes, untreated fibroblasts; closed boxes, TGF-β-treated fibroblasts. *p<0.005. C. MEFs were transfected with pEBS 4 -luc inpresence or absence of Nab2. Following 24 h incubation, cultures were harvested and cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase, are the means±S.D. of triplicate determinations. D. siRNA knockdown. Normal dermal fibroblasts were transfected with Nab2 siRNA or irrelevant negative control siRNA, and incubated with TGF-β. Twenty-four h later, fibroblasts were harvested. Levels of Nab2 and collagen were determined by Western analysis of whole cell lysates.

Article Snippet: Immunoprecipitated proteins were subjected to Western analysis as above, using primary antibodies specific for HDAC1 (C19) or Nab2 (IC4) (both from Santa Cruz).

Techniques: Cell Culture, Incubation, Western Blot, Transfection, Luciferase, Negative Control

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Skin from Nab2 −/− mice and wildtype mice was harvested. A. Tissues were stained with hematoxylin and eosin (a–d) or picrosirius red (e,f). Original magnification ×100 (a, b), ×400 (c–f). B. Immunofluorescence using antibodies to α-smooth muscle actin (green). Nuclei were identified by DAPI (blue). Original magnification ×100 (a, d), ×200 (b, c, e, f). C. Total RNA was harvested and was subjected to real-time qPCR analysis. Results, expressed relative to 18S, are the means±S.D. of triplicate determinations from a representative experiment.

Article Snippet: Immunoprecipitated proteins were subjected to Western analysis as above, using primary antibodies specific for HDAC1 (C19) or Nab2 (IC4) (both from Santa Cruz).

Techniques: Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: A. Foreskin fibroblasts transfected with Nab2 were incubated with TGF-β1 for 24 h. Whole cell lysates were immunoprecipitated with antibodies against Egr-1 and immunoblotted using indicated antibodies. Representative immunoblots. B. ChIP assays. Fibroblasts transfected with Nab2 were formaldehyde cross-linked and immunoprecipitated with indicated antibodies, followed by amplification of the captured DNA using COL1A2-specific primers. Input genomic DNA was used as positive control. C. Fibroblasts transfected with Nab2 were incubated with TGF-β for 2 h. Cells were then fixed, incubated with indicated antibodies and examined by immunofluorescence confocal microscopy. Nuclei were identified by DAPI (blue). Representative images. Original magnification ×400. D. NIH3T3 fibroblasts were cotransfected with expression vectors for Egr-1, Nab2, wildtype CHD4 or mutant CHD4 del(1–1280) or empty vector, along with pEBS 4 -luc. Cultures were harvested 24 h later, and cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase, are the means±S.D. of triplicate determinations. *p<0.005. E. Cartoon illustrating the mechanism underlying modulation of collagen gene expression by Nab2. In unstimulated fibroblasts (basal state), small amounts of Egr-1 and Nab2 are constitutively associated with the COL1A2 promoter. Upon transient TGF-β stimulation, Egr-1 is induced and recruited to the COL1A2 promoter, where together with p300 it enhances histone H4 hyperacetylation and stimulates transcription. More sustained TGF-β stimulation leads to increased Nab2 expression and its accumulation in the Egr-1-COL1A2 transcriptional complex, with HDAC1 recruitment, H4 deacetylation, and transcriptional repression. In scleroderma fibroblasts, defective Nab2 induction or function might results in unopposed Egr-1 signaling and target gene transcription. For full explanation, see text .

Article Snippet: Immunoprecipitated proteins were subjected to Western analysis as above, using primary antibodies specific for HDAC1 (C19) or Nab2 (IC4) (both from Santa Cruz).

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Amplification, Positive Control, Immunofluorescence, Confocal Microscopy, Expressing, Mutagenesis, Plasmid Preparation, Luciferase

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Lesional skin biopsies were obtained from patients with scleroderma (n = 6) and healthy controls (n = 3), and processed for immunohistochemistry as described under . A. Nab2 expression. Skin biopsies from healthy controls (a, b, e) and scleroderma patients (c, d, f, g, h–l). Original magnification ×100(a, h), ×400 (b, d, i), ×630 (c, e, f, j, k, l). Note uniformly intense Nab2 nuclear immunoreactivity in epithelial cells in scleroderma skin biopsies (h, i, l), as well as in perifollicular and periglandular (c, d, g) epithelial cells and vascular endothelial cells (f, j), compared to weak and variable Nab2 expression seen in occasional dermal fibroblasts (k, l). Representative photomicrographs; similar immunostaining pattern was observed in all six scleroderma skin biopsies. B. Phospho-Smad2 (a–d) or Egr-1 (e–h) expression. Nuclei are counterstained with hematoxylin (blue). Note strong immunostaining in dermal fibroblasts in scleroderma (d, h) skin biopsies compared to healthy controls (b, f).

Article Snippet: Immunoprecipitated proteins were subjected to Western analysis as above, using primary antibodies specific for HDAC1 (C19) or Nab2 (IC4) (both from Santa Cruz).

Techniques: Immunohistochemistry, Expressing, Immunostaining

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Oligonucleotides used for Real-Time Quantitative PCR.

Article Snippet: Cultures were then fixed and permeabilized with paraformaldehyde and 100% methanol, and incubated with antibodies to Nab2 (Santa Cruz) or to Type I collagen (Southern Biotechnology) at a 1∶100 dilution, followed by Alexa-fluor secondary antibodies (Invitrogen).

Techniques:

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Confluent cultures of human foreskin fibroblasts were incubated with TGF-β1 (10 ng/ml) for indicated periods. A, D Total RNA was isolated and analysed by real-time quantitative PCR. Results, normalized with actin, are the means±S.D. of triplicate determinations from a representative experiment. B. Whole cell lysates were subjected to Western analysis. Representative immunoblots. C. Fibroblasts were fixed and stained with anti-Nab2 antibodies, or DAPI to detect nuclei, and viewed by immunofluorescence microscopy (original magnification ×100).

Article Snippet: Cultures were then fixed and permeabilized with paraformaldehyde and 100% methanol, and incubated with antibodies to Nab2 (Santa Cruz) or to Type I collagen (Southern Biotechnology) at a 1∶100 dilution, followed by Alexa-fluor secondary antibodies (Invitrogen).

Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Staining, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Cultures of fibroblasts were cotransfected with Nab2 expression vectors or empty vector and harvested following incubation with TGF-β1 for 24 or 48 h. A. Whole cell lysates were examined by Western analysis. Representative immunoblots. B. Fibroblasts were fixed, incubated with antibodies to Type I collagen, and examined by immunofluorescence microscopy (original magnification ×400). Nuclei were identified by DAPI (blue). C. Total RNA was isolated and examined by real-time qPCR. Results, normalized with actin, are the means±S.D. of triplicate determinations from a representative experiment. D. Fibroblasts were cotransfected with 772COL1A2-CAT. Cell lysates were assayed for their CAT activities (upper panels) or examined by Western analysis (lower panels). Results of CAT assays, normalized with Renilla luciferase, are expressed as means±S.D. of triplicate determinations from a representative experiment. Open boxes, untreated fibroblasts; closed boxes, TGF-β-treated fibroblasts. *p<0.005. E. After 48 h fibroblasts were fixed, incubated with antibodies to α-SMA (green) and examined by immunofluorescence microscopy. Nuclei were identified by DAPI (blue). Representative images (original magnification ×100).

Article Snippet: Cultures were then fixed and permeabilized with paraformaldehyde and 100% methanol, and incubated with antibodies to Nab2 (Santa Cruz) or to Type I collagen (Southern Biotechnology) at a 1∶100 dilution, followed by Alexa-fluor secondary antibodies (Invitrogen).

Techniques: Expressing, Plasmid Preparation, Incubation, Western Blot, Immunofluorescence, Microscopy, Isolation, Luciferase

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Wildtype and Nab2 −/− mouse embryonic fibroblasts (MEFs) cultured in parallel were incubated with or without TGF-β1 for 24 h. A. Whole cell lysates and culture supernatants were subjected to Western analysis. Representative immunoblots. B. Total RNA was subjected to real-time qPCR analysis. Results, expressed relative to 18 s, are the means±S.D. of triplicate determinations from a representative experiment. Open boxes, untreated fibroblasts; closed boxes, TGF-β-treated fibroblasts. *p<0.005. C. MEFs were transfected with pEBS 4 -luc inpresence or absence of Nab2. Following 24 h incubation, cultures were harvested and cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase, are the means±S.D. of triplicate determinations. D. siRNA knockdown. Normal dermal fibroblasts were transfected with Nab2 siRNA or irrelevant negative control siRNA, and incubated with TGF-β. Twenty-four h later, fibroblasts were harvested. Levels of Nab2 and collagen were determined by Western analysis of whole cell lysates.

Article Snippet: Cultures were then fixed and permeabilized with paraformaldehyde and 100% methanol, and incubated with antibodies to Nab2 (Santa Cruz) or to Type I collagen (Southern Biotechnology) at a 1∶100 dilution, followed by Alexa-fluor secondary antibodies (Invitrogen).

Techniques: Cell Culture, Incubation, Western Blot, Transfection, Luciferase, Negative Control

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Skin from Nab2 −/− mice and wildtype mice was harvested. A. Tissues were stained with hematoxylin and eosin (a–d) or picrosirius red (e,f). Original magnification ×100 (a, b), ×400 (c–f). B. Immunofluorescence using antibodies to α-smooth muscle actin (green). Nuclei were identified by DAPI (blue). Original magnification ×100 (a, d), ×200 (b, c, e, f). C. Total RNA was harvested and was subjected to real-time qPCR analysis. Results, expressed relative to 18S, are the means±S.D. of triplicate determinations from a representative experiment.

Article Snippet: Cultures were then fixed and permeabilized with paraformaldehyde and 100% methanol, and incubated with antibodies to Nab2 (Santa Cruz) or to Type I collagen (Southern Biotechnology) at a 1∶100 dilution, followed by Alexa-fluor secondary antibodies (Invitrogen).

Techniques: Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: A. Foreskin fibroblasts transfected with Nab2 were incubated with TGF-β1 for 24 h. Whole cell lysates were immunoprecipitated with antibodies against Egr-1 and immunoblotted using indicated antibodies. Representative immunoblots. B. ChIP assays. Fibroblasts transfected with Nab2 were formaldehyde cross-linked and immunoprecipitated with indicated antibodies, followed by amplification of the captured DNA using COL1A2-specific primers. Input genomic DNA was used as positive control. C. Fibroblasts transfected with Nab2 were incubated with TGF-β for 2 h. Cells were then fixed, incubated with indicated antibodies and examined by immunofluorescence confocal microscopy. Nuclei were identified by DAPI (blue). Representative images. Original magnification ×400. D. NIH3T3 fibroblasts were cotransfected with expression vectors for Egr-1, Nab2, wildtype CHD4 or mutant CHD4 del(1–1280) or empty vector, along with pEBS 4 -luc. Cultures were harvested 24 h later, and cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase, are the means±S.D. of triplicate determinations. *p<0.005. E. Cartoon illustrating the mechanism underlying modulation of collagen gene expression by Nab2. In unstimulated fibroblasts (basal state), small amounts of Egr-1 and Nab2 are constitutively associated with the COL1A2 promoter. Upon transient TGF-β stimulation, Egr-1 is induced and recruited to the COL1A2 promoter, where together with p300 it enhances histone H4 hyperacetylation and stimulates transcription. More sustained TGF-β stimulation leads to increased Nab2 expression and its accumulation in the Egr-1-COL1A2 transcriptional complex, with HDAC1 recruitment, H4 deacetylation, and transcriptional repression. In scleroderma fibroblasts, defective Nab2 induction or function might results in unopposed Egr-1 signaling and target gene transcription. For full explanation, see text .

Article Snippet: Cultures were then fixed and permeabilized with paraformaldehyde and 100% methanol, and incubated with antibodies to Nab2 (Santa Cruz) or to Type I collagen (Southern Biotechnology) at a 1∶100 dilution, followed by Alexa-fluor secondary antibodies (Invitrogen).

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Amplification, Positive Control, Immunofluorescence, Confocal Microscopy, Expressing, Mutagenesis, Plasmid Preparation, Luciferase

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Lesional skin biopsies were obtained from patients with scleroderma (n = 6) and healthy controls (n = 3), and processed for immunohistochemistry as described under . A. Nab2 expression. Skin biopsies from healthy controls (a, b, e) and scleroderma patients (c, d, f, g, h–l). Original magnification ×100(a, h), ×400 (b, d, i), ×630 (c, e, f, j, k, l). Note uniformly intense Nab2 nuclear immunoreactivity in epithelial cells in scleroderma skin biopsies (h, i, l), as well as in perifollicular and periglandular (c, d, g) epithelial cells and vascular endothelial cells (f, j), compared to weak and variable Nab2 expression seen in occasional dermal fibroblasts (k, l). Representative photomicrographs; similar immunostaining pattern was observed in all six scleroderma skin biopsies. B. Phospho-Smad2 (a–d) or Egr-1 (e–h) expression. Nuclei are counterstained with hematoxylin (blue). Note strong immunostaining in dermal fibroblasts in scleroderma (d, h) skin biopsies compared to healthy controls (b, f).

Article Snippet: Cultures were then fixed and permeabilized with paraformaldehyde and 100% methanol, and incubated with antibodies to Nab2 (Santa Cruz) or to Type I collagen (Southern Biotechnology) at a 1∶100 dilution, followed by Alexa-fluor secondary antibodies (Invitrogen).

Techniques: Immunohistochemistry, Expressing, Immunostaining

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Oligonucleotides used for Real-Time Quantitative PCR.

Article Snippet: Following incubation of the slides using primary antibodies against anti-mouse Nab2 monoclonal antibodies (Santa Cruz, 1∶50 dilution), anti-rabbit phospho-Smad2 (Cell Signaling Technology, 1∶100 dilution), and anti-rabbit Egr-1 (Santa Cruz, 1∶100 dilution), appropriate goat anti-mouse IgG or donkey anti goat IgG (Dako) were applied as secondary antibodies.

Techniques:

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Confluent cultures of human foreskin fibroblasts were incubated with TGF-β1 (10 ng/ml) for indicated periods. A, D Total RNA was isolated and analysed by real-time quantitative PCR. Results, normalized with actin, are the means±S.D. of triplicate determinations from a representative experiment. B. Whole cell lysates were subjected to Western analysis. Representative immunoblots. C. Fibroblasts were fixed and stained with anti-Nab2 antibodies, or DAPI to detect nuclei, and viewed by immunofluorescence microscopy (original magnification ×100).

Article Snippet: Following incubation of the slides using primary antibodies against anti-mouse Nab2 monoclonal antibodies (Santa Cruz, 1∶50 dilution), anti-rabbit phospho-Smad2 (Cell Signaling Technology, 1∶100 dilution), and anti-rabbit Egr-1 (Santa Cruz, 1∶100 dilution), appropriate goat anti-mouse IgG or donkey anti goat IgG (Dako) were applied as secondary antibodies.

Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Staining, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Cultures of fibroblasts were cotransfected with Nab2 expression vectors or empty vector and harvested following incubation with TGF-β1 for 24 or 48 h. A. Whole cell lysates were examined by Western analysis. Representative immunoblots. B. Fibroblasts were fixed, incubated with antibodies to Type I collagen, and examined by immunofluorescence microscopy (original magnification ×400). Nuclei were identified by DAPI (blue). C. Total RNA was isolated and examined by real-time qPCR. Results, normalized with actin, are the means±S.D. of triplicate determinations from a representative experiment. D. Fibroblasts were cotransfected with 772COL1A2-CAT. Cell lysates were assayed for their CAT activities (upper panels) or examined by Western analysis (lower panels). Results of CAT assays, normalized with Renilla luciferase, are expressed as means±S.D. of triplicate determinations from a representative experiment. Open boxes, untreated fibroblasts; closed boxes, TGF-β-treated fibroblasts. *p<0.005. E. After 48 h fibroblasts were fixed, incubated with antibodies to α-SMA (green) and examined by immunofluorescence microscopy. Nuclei were identified by DAPI (blue). Representative images (original magnification ×100).

Article Snippet: Following incubation of the slides using primary antibodies against anti-mouse Nab2 monoclonal antibodies (Santa Cruz, 1∶50 dilution), anti-rabbit phospho-Smad2 (Cell Signaling Technology, 1∶100 dilution), and anti-rabbit Egr-1 (Santa Cruz, 1∶100 dilution), appropriate goat anti-mouse IgG or donkey anti goat IgG (Dako) were applied as secondary antibodies.

Techniques: Expressing, Plasmid Preparation, Incubation, Western Blot, Immunofluorescence, Microscopy, Isolation, Luciferase

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Wildtype and Nab2 −/− mouse embryonic fibroblasts (MEFs) cultured in parallel were incubated with or without TGF-β1 for 24 h. A. Whole cell lysates and culture supernatants were subjected to Western analysis. Representative immunoblots. B. Total RNA was subjected to real-time qPCR analysis. Results, expressed relative to 18 s, are the means±S.D. of triplicate determinations from a representative experiment. Open boxes, untreated fibroblasts; closed boxes, TGF-β-treated fibroblasts. *p<0.005. C. MEFs were transfected with pEBS 4 -luc inpresence or absence of Nab2. Following 24 h incubation, cultures were harvested and cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase, are the means±S.D. of triplicate determinations. D. siRNA knockdown. Normal dermal fibroblasts were transfected with Nab2 siRNA or irrelevant negative control siRNA, and incubated with TGF-β. Twenty-four h later, fibroblasts were harvested. Levels of Nab2 and collagen were determined by Western analysis of whole cell lysates.

Article Snippet: Following incubation of the slides using primary antibodies against anti-mouse Nab2 monoclonal antibodies (Santa Cruz, 1∶50 dilution), anti-rabbit phospho-Smad2 (Cell Signaling Technology, 1∶100 dilution), and anti-rabbit Egr-1 (Santa Cruz, 1∶100 dilution), appropriate goat anti-mouse IgG or donkey anti goat IgG (Dako) were applied as secondary antibodies.

Techniques: Cell Culture, Incubation, Western Blot, Transfection, Luciferase, Negative Control

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Skin from Nab2 −/− mice and wildtype mice was harvested. A. Tissues were stained with hematoxylin and eosin (a–d) or picrosirius red (e,f). Original magnification ×100 (a, b), ×400 (c–f). B. Immunofluorescence using antibodies to α-smooth muscle actin (green). Nuclei were identified by DAPI (blue). Original magnification ×100 (a, d), ×200 (b, c, e, f). C. Total RNA was harvested and was subjected to real-time qPCR analysis. Results, expressed relative to 18S, are the means±S.D. of triplicate determinations from a representative experiment.

Article Snippet: Following incubation of the slides using primary antibodies against anti-mouse Nab2 monoclonal antibodies (Santa Cruz, 1∶50 dilution), anti-rabbit phospho-Smad2 (Cell Signaling Technology, 1∶100 dilution), and anti-rabbit Egr-1 (Santa Cruz, 1∶100 dilution), appropriate goat anti-mouse IgG or donkey anti goat IgG (Dako) were applied as secondary antibodies.

Techniques: Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: A. Foreskin fibroblasts transfected with Nab2 were incubated with TGF-β1 for 24 h. Whole cell lysates were immunoprecipitated with antibodies against Egr-1 and immunoblotted using indicated antibodies. Representative immunoblots. B. ChIP assays. Fibroblasts transfected with Nab2 were formaldehyde cross-linked and immunoprecipitated with indicated antibodies, followed by amplification of the captured DNA using COL1A2-specific primers. Input genomic DNA was used as positive control. C. Fibroblasts transfected with Nab2 were incubated with TGF-β for 2 h. Cells were then fixed, incubated with indicated antibodies and examined by immunofluorescence confocal microscopy. Nuclei were identified by DAPI (blue). Representative images. Original magnification ×400. D. NIH3T3 fibroblasts were cotransfected with expression vectors for Egr-1, Nab2, wildtype CHD4 or mutant CHD4 del(1–1280) or empty vector, along with pEBS 4 -luc. Cultures were harvested 24 h later, and cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase, are the means±S.D. of triplicate determinations. *p<0.005. E. Cartoon illustrating the mechanism underlying modulation of collagen gene expression by Nab2. In unstimulated fibroblasts (basal state), small amounts of Egr-1 and Nab2 are constitutively associated with the COL1A2 promoter. Upon transient TGF-β stimulation, Egr-1 is induced and recruited to the COL1A2 promoter, where together with p300 it enhances histone H4 hyperacetylation and stimulates transcription. More sustained TGF-β stimulation leads to increased Nab2 expression and its accumulation in the Egr-1-COL1A2 transcriptional complex, with HDAC1 recruitment, H4 deacetylation, and transcriptional repression. In scleroderma fibroblasts, defective Nab2 induction or function might results in unopposed Egr-1 signaling and target gene transcription. For full explanation, see text .

Article Snippet: Following incubation of the slides using primary antibodies against anti-mouse Nab2 monoclonal antibodies (Santa Cruz, 1∶50 dilution), anti-rabbit phospho-Smad2 (Cell Signaling Technology, 1∶100 dilution), and anti-rabbit Egr-1 (Santa Cruz, 1∶100 dilution), appropriate goat anti-mouse IgG or donkey anti goat IgG (Dako) were applied as secondary antibodies.

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Amplification, Positive Control, Immunofluorescence, Confocal Microscopy, Expressing, Mutagenesis, Plasmid Preparation, Luciferase

Journal: PLoS ONE

Article Title: The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

doi: 10.1371/journal.pone.0007620

Figure Lengend Snippet: Lesional skin biopsies were obtained from patients with scleroderma (n = 6) and healthy controls (n = 3), and processed for immunohistochemistry as described under . A. Nab2 expression. Skin biopsies from healthy controls (a, b, e) and scleroderma patients (c, d, f, g, h–l). Original magnification ×100(a, h), ×400 (b, d, i), ×630 (c, e, f, j, k, l). Note uniformly intense Nab2 nuclear immunoreactivity in epithelial cells in scleroderma skin biopsies (h, i, l), as well as in perifollicular and periglandular (c, d, g) epithelial cells and vascular endothelial cells (f, j), compared to weak and variable Nab2 expression seen in occasional dermal fibroblasts (k, l). Representative photomicrographs; similar immunostaining pattern was observed in all six scleroderma skin biopsies. B. Phospho-Smad2 (a–d) or Egr-1 (e–h) expression. Nuclei are counterstained with hematoxylin (blue). Note strong immunostaining in dermal fibroblasts in scleroderma (d, h) skin biopsies compared to healthy controls (b, f).

Article Snippet: Following incubation of the slides using primary antibodies against anti-mouse Nab2 monoclonal antibodies (Santa Cruz, 1∶50 dilution), anti-rabbit phospho-Smad2 (Cell Signaling Technology, 1∶100 dilution), and anti-rabbit Egr-1 (Santa Cruz, 1∶100 dilution), appropriate goat anti-mouse IgG or donkey anti goat IgG (Dako) were applied as secondary antibodies.

Techniques: Immunohistochemistry, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Inducible CRISPR epigenome systems mimic cocaine induced bidirectional regulation of Nab2 and Egr3

doi: 10.1101/2022.09.19.508525

Figure Lengend Snippet: Nab2 ribosome-associated mRNA expression after repeated cocaine exposure. A . Male mice received intraperitoneal injection of cocaine (20mg/kg) for 7 consecutive days. NAc punches were harvested 24 hours post last cocaine injection. B . qRT-PCR analysis shows a trend towards an increase in Nab2 mRNA expression in the NAc after repeated cocaine exposure in male mice (saline n= 6, cocaine n= 7). C . Transcription repression associated H3K9me2 and H3K27me3 marks show bidirectional enrichment on Nab2 promoter after repeated exposure to cocaine. A transcription activation associated H3K4me3 mark and a histone demethylase Kdm1a both showed an increased enrichment on Nab2 promoter after repeated exposure to cocaine (H3K4me3 saline n= 6, cocaine n= 6; H3K9me2 saline n= 6, cocaine n= 5; H3K27me3 saline n= 6, cocaine n= 5; Kdm1a saline n= 6, cocaine n= 6). D . D1-Cre-RT and D2-Cre-RT mice allow cell subtype specific isolation of ribosome associated mRNA. E . Repeated cocaine exposure reduced Nab2 mRNA expression in D1-MSNs while inducing Nab2 mRNA expression in D2-MSNs (D1-MSNs saline n= 11, cocaine n= 8; D2-MSNs saline n= 9, cocaine n= 8). Each bar represents mean ± SEM. * p <0.05, ** p <0.01.

Article Snippet: Before sample sonication, IgG magnetic beads (Invitrogen; Sheep anti-rabbit 11202D) were incubated in blocking solution (0.5% BSA in PBS) which contains 15ug of anti-Nab2 antibody (Santa Cruz SC22815) per reaction at 4°C overnight under constant rotation.

Techniques: Expressing, Injection, Quantitative RT-PCR, Activation Assay, Isolation

Journal: bioRxiv

Article Title: Inducible CRISPR epigenome systems mimic cocaine induced bidirectional regulation of Nab2 and Egr3

doi: 10.1101/2022.09.19.508525

Figure Lengend Snippet: CRISPRi and CRISPRa manipulation of Nab2 and Egr3 transcription. A . qRT-PCR shows Nab2 targeted CRISPRi repressed Nab2 mRNA while inducing Egr3 mRNA expression, and Egr3 targeted CRISPRi repressed Egr3 mRNA while inducing Nab2 mRNA expression in Neuro2a cells (n= 9 per condition). B . Conversely, Nab2 targeted CRISPRa activates Nab2 mRNA expression, and Egr3 targeted CRISPRa activates Egr3 mRNA expression in Neuro2a cells (n= 9 per condition). C . Western blots using Neuro2a cells transfected with Nab2 targeted CRISPRi show downregulation of NAB2 and upregulation of EGR3, while cells transfected with Nab2 targeted CRISPRa show upregulation of NAB2 and downregulation of EGR3. Consistently, Neuro2a cells transfected with Egr3 targeted CRISPRi samples show downregulation of EGR3 and upregulation of NAB2, while cells transfected with Egr3 targeted CRISPRa show upregulation of EGR3 but did not change NAB2 levels (n= 3 per condition). D . Cut and Run of Neuro2a cells transfected with CRISPRi show HA-tag enrichment on the promoters of respective genes targeted by sgRNAs. Transcriptional activation marks, H3K4me3 and H3K27ac, display reduced enrichment on the promoters of respective genes targeted by sgRNAs compared to lacZ controls (n= 7-9 per condition). E . Cut and Run of Neuro2a cells transfected with CRISPRa show HA-tag, H3K4me3, and H3K27ac enrichment on the promoters of respective genes targeted by sgRNAs compared to lacZ controls (n= 9 per condition). Each bar represents mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: Before sample sonication, IgG magnetic beads (Invitrogen; Sheep anti-rabbit 11202D) were incubated in blocking solution (0.5% BSA in PBS) which contains 15ug of anti-Nab2 antibody (Santa Cruz SC22815) per reaction at 4°C overnight under constant rotation.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Activation Assay

Journal: bioRxiv

Article Title: Inducible CRISPR epigenome systems mimic cocaine induced bidirectional regulation of Nab2 and Egr3

doi: 10.1101/2022.09.19.508525

Figure Lengend Snippet: Development of a Cre and light inducible Opto-CRISPR-KDM1A system. A . Illustration of light inducible dCas9 and KDM1A fusion complex using CIBN and CRY2 heterodimers. Neuro2a cells received 2 hours of 1mW blue light stimulation. B . Immunocytochemistry of Neuro2a cells transfected with Opto-CRISPR-KDM1A display a Cre inducible expression as observed by GFP (green), HA-Tag (red), and Flag-Tag (magenta) expression. DAPI signal is blue. Scale bar, 50μM. C . qRT-PCR demonstrates cells transfected only with DIO-CRY2-FLAG-KDM1Aand Cre vectors do not show significant changes in Nab2 or Egr3 mRNA levels compared to cells transfected with complete Opto-CRISPR-KDM1A targeted by lacZ sgRNA after 2 hours of blue light stimulation (n= 3 per condition). D . qRT-PCR demonstrates that Neuro2a cells transfected with Opto-CRISPR-KDM1A with lacZ sgRNA in no light condition and 2 hours of 1mW blue light stimulation do not display significant differences in Nab2 and Egr3 mRNA levels (n= 3 per condition). Each bar represents mean ± SEM. n.s., not significant.

Article Snippet: Before sample sonication, IgG magnetic beads (Invitrogen; Sheep anti-rabbit 11202D) were incubated in blocking solution (0.5% BSA in PBS) which contains 15ug of anti-Nab2 antibody (Santa Cruz SC22815) per reaction at 4°C overnight under constant rotation.

Techniques: CRISPR, Immunocytochemistry, Transfection, Expressing, FLAG-tag, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Inducible CRISPR epigenome systems mimic cocaine induced bidirectional regulation of Nab2 and Egr3

doi: 10.1101/2022.09.19.508525

Figure Lengend Snippet: Opto-CRISPR-KDM1A mediated inhibition of Nab2 and Egr3 transcription. A . qRT-PCR demonstrates that 2 hours of blue light stimulation downregulated Nab2 mRNA while inducing Egr3 mRNA expression in cells transfected with Nab2 targeted Opto-CRISPR-KDM1A. Conversely, 2 hours of blue light stimulation induced Nab2 mRNA while downregulating Egr3 mRNA expression in cells transfected with Opto-CRISPR-KDM1A and Egr3 sgRNA (n= 9 per condition). B . Western blots show 2 hours of blue light stimulation downregulated NAB2 levels while upregulating EGR3 levels in cells transfected Opto-CRISPR-KDM1A and Nab2 sgRNA. EGR3 levels were downregulated while NAB2 levels were unchanged in cells transfected with Opto-CRISPR-KDM1A and Egr3 sgRNA (n= 3 per condition). C . Cut and Run on blue light stimulated Neuro2a cells transfected with Opto-CRISPR-KDM1A shows enrichment of Opto-CRISPR-KDM1A complex on the promoters of respective genes targeted by sgRNAs compared to lacZ sgRNA controls. Transcriptional activation marks, H3K4me3 and H3K27ac, have reduced enrichment on the promoters of respective genes targeted by sgRNAs compared to lacZ sgRNA controls (n= 9 per condition). Each bar represents mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: Before sample sonication, IgG magnetic beads (Invitrogen; Sheep anti-rabbit 11202D) were incubated in blocking solution (0.5% BSA in PBS) which contains 15ug of anti-Nab2 antibody (Santa Cruz SC22815) per reaction at 4°C overnight under constant rotation.

Techniques: CRISPR, Inhibition, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: Inducible CRISPR epigenome systems mimic cocaine induced bidirectional regulation of Nab2 and Egr3

doi: 10.1101/2022.09.19.508525

Figure Lengend Snippet: Development of a Cre and light inducible Opto-CRISPR-p300 system. A . Illustration of light inducible dCas9 and p300 fusion complex using CIBN and CRY2 heterodimers. Neuro2a cells received 2 hours of 1mW blue light stimulation. B . Immunocytochemistry of Neuro2a cells transfected with Opto-CRISPR-p300 system demonstrate a Cre inducible expression as observed by GFP (green), HA-Tag (red), and Flag-Tag (magenta) expression. DAPI signal is blue. Scale bar, 50μM. C . qRT-PCR demonstrates that the expression of DIO-CRY2-FLAG-p300core alone does not induce changes in Nab2 or Egr3 mRNA levels compared to cells transfected with Opto-CRISPR-p300 targeted by lacZ sgRNA after 2 hours of blue light stimulation (n= 3 per condition). D . qRT-PCR demonstrates Neuro2a cells transfected with Opto-CRISPR-p300 targeted by lacZ sgRNA in no light condition and 2 hours of 1mW blue light stimulation do not display significant differences in Nab2 and Egr3 mRNA levels (n= 3 per condition). Each bar represents mean ± SEM. n.s., not significant.

Article Snippet: Before sample sonication, IgG magnetic beads (Invitrogen; Sheep anti-rabbit 11202D) were incubated in blocking solution (0.5% BSA in PBS) which contains 15ug of anti-Nab2 antibody (Santa Cruz SC22815) per reaction at 4°C overnight under constant rotation.

Techniques: CRISPR, Immunocytochemistry, Transfection, Expressing, FLAG-tag, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Inducible CRISPR epigenome systems mimic cocaine induced bidirectional regulation of Nab2 and Egr3

doi: 10.1101/2022.09.19.508525

Figure Lengend Snippet: Opto-CRISPR-p300 mediated activation of Nab2 and Egr3 transcription. A . qRT-PCR demonstrate that 2 hours of blue light stimulation induced Nab2 mRNA and Egr3 mRNA expressios in cells transfected with Nab2 or Egr3 sgRNAs respectively and Opto-CRISPR-p300 (n= 9 per condition). B . Western blots demonstrate that 2 hours of blue light stimulation induced NAB2 levels while downregulating EGR3 levels in cells transfected with Opto-CRISPR-p300 and Nab2 sgRNA. EGR3 levels were upregulated while NAB2 levels were unchanged in cells transfected with Opto-CRISPR-p300 and Egr3 sgRNA (n= 3 per condition). C . Cut and Run of blue light stimulated Neuro2a cells transfected with Opto-CRISPR-p300 display enrichment of Opto-CRISPR-p300 complex on the promoters of respective genes targeted by sgRNAs compared to lacZ sgRNA controls. Transcriptional activation marks, H3K4me3 and H3K27ac, have increased enrichment on the promoters of respective genes targeted by sgRNAs compared to lacZ sgRNA controls (n= 9 per condition). Each bar represents mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: Before sample sonication, IgG magnetic beads (Invitrogen; Sheep anti-rabbit 11202D) were incubated in blocking solution (0.5% BSA in PBS) which contains 15ug of anti-Nab2 antibody (Santa Cruz SC22815) per reaction at 4°C overnight under constant rotation.

Techniques: CRISPR, Activation Assay, Quantitative RT-PCR, Transfection, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Serine hydroxymethyltransferase 2 expression promotes tumorigenesis in rhabdomyosarcoma with 12q13-q14 amplification

doi: 10.1172/JCI138022

Figure Lengend Snippet: Cell lines are indicated by numbers: FN: 1-Rh6, 2-RD, 3-SMS-CTR; FP: 4-CW9019, 5-MP4, 6-RMS-097, 7-Rh3, 8-Rh5, 9-Rh28, 10-Rh41 and 11-Rh30 (all were PAX3-FOXO1-positive except for CW9019, which is PAX7-FOXO1-positive). (A) Screening for the 12q13-q14 amplicon in RMS cell lines. Graph shows relative copy numbers of CDK4 and SHMT2 in genomic DNA quantified by qPCR and normalized against the centromeric D12Z3. RD cells were used as a negative control in which copy numbers of CDK4 and SHMT2 were set at 1.0. Data are represented as mean ± SD of 6 technical replicates from 2 biological replicates. Statistical analysis of copy numbers in Rh30 versus the other lines was performed using Student’s t test. *P < 0.01. (B) mRNA expression of NEMP1, NAB2, SHMT2, and R3HDM2. Graph shows relative mRNA expression quantified by quantitative reverse-transcriptase PCR (qRT-PCR). GAPDH was used for normalization. Data are represented as mean ± SD of 4 replicates. Statistical analysis of expression differences in Rh30 versus the other lines was performed using Student’s t test. *P < 0.05. (C) Protein expression of SHMT2, NAB2, FOXO1, and PAX3/7-FOXO1 in cell lines. The immunoblot was probed with SHMT2 antibody, stripped, and reprobed with other antibodies. Relevant areas of the blot are shown. GAPDH was used as a loading control.

Article Snippet: Membranes were incubated overnight with antibodies against SHMT1 (1:1000, catalog 80715, Cell Signaling Technology), SHMT2 (1:1000, catalog 12762, Cell Signaling Technology), NAB2 (1:200, catalog sc-23867, Santa Cruz Biotechnology Inc.), and GAPDH (1:200, catalog sc-47724, Santa Cruz Biotechnology Inc.).

Techniques: Amplification, Negative Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Differential Interleukin-2 Transcription Kinetics Render Mouse but Not Human T Cells Vulnerable to Splicing Inhibition Early after Activation

doi: 10.1128/MCB.00035-19

Figure Lengend Snippet: Role of Egr1 and Nab2 in mediating the sensitivity to splicing inhibition. (A) Temporal gene expression analysis in EL4 cells using RNA-Seq of chromatin-associated RNA; (B and C) temporal RNA level (mean ± SD; n = 3) (B) and protein level (C) of Egr1 and Nab2 in EL4 and Jurkat cells; (D) siRNA knockdown efficiency for Egr1 and Nab2 in EL4 cells (mean ± SD; n = 3); (E) cell viability analysis in Egr1 and Nab2 knockdown EL4 cells at 48 h poststimulation (relative to nonstimulated [NS] cells) (mean ± SD; n = 3); (F) temporal splicing modulation analysis (with Pld B) in siRNA-transfected EL4 cells. **, P < 0.001 (Student's unpaired t test, done using GraphPad software).

Article Snippet: The antibodies used for Western blotting were as follows: anti-Egr-1 (sc-515830; Santa Cruz), anti-Nab2 (sc-23867; Santa Cruz), and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; GT239; GeneTex).

Techniques: Inhibition, Expressing, RNA Sequencing Assay, Transfection, Software

Journal: Molecular and Cellular Biology

Article Title: Differential Interleukin-2 Transcription Kinetics Render Mouse but Not Human T Cells Vulnerable to Splicing Inhibition Early after Activation

doi: 10.1128/MCB.00035-19

Figure Lengend Snippet: IL-2 mediates the sensitivity to splicing inhibition sensitivity. (A) Relative IL-2 expression in the presence and absence of Nab2 (mean ± SD; n = 3); (B) temporal IL-2 total mRNA level in EL4 and Jurkat cells (mean ± SD; n = 3); (C) IL-2 de novo transcription dynamics in EL4 and Jurkat cells (mean ± SD; n = 3); (D) siRNA knockdown efficiency for IL-2 in EL4 cells (mean ± SD; n = 3); (E) cell viability analysis in IL-2 knockdown EL4 cells at 48 h poststimulation (relative to that in nonstimulated cells) (mean ± SD; n = 3); (F) temporal splicing modulation analysis (with Pld B) in siRNA-transfected EL4 cells; (G) temporal splicing modulation analysis in mouse primary T cells in the presence of growth medium supplemented or not supplemented with IL-2. We used the data from the assay whose results are presented in Fig. 1C for direct comparison of conditions with or without IL-2 supplementation. Experiments were performed in parallel. *, P < 0.01; ***, P < 0.0001 (Student's unpaired t test, done using GraphPad software).

Article Snippet: The antibodies used for Western blotting were as follows: anti-Egr-1 (sc-515830; Santa Cruz), anti-Nab2 (sc-23867; Santa Cruz), and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; GT239; GeneTex).

Techniques: Inhibition, Expressing, Transfection, Software

Journal: Molecular and Cellular Biology

Article Title: Differential Interleukin-2 Transcription Kinetics Render Mouse but Not Human T Cells Vulnerable to Splicing Inhibition Early after Activation

doi: 10.1128/MCB.00035-19

Figure Lengend Snippet: siRNAs

Article Snippet: The antibodies used for Western blotting were as follows: anti-Egr-1 (sc-515830; Santa Cruz), anti-Nab2 (sc-23867; Santa Cruz), and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; GT239; GeneTex).

Techniques: Sequencing

Journal: Molecular and Cellular Biology

Article Title: Differential Interleukin-2 Transcription Kinetics Render Mouse but Not Human T Cells Vulnerable to Splicing Inhibition Early after Activation

doi: 10.1128/MCB.00035-19

Figure Lengend Snippet: Primers for qPCR

Article Snippet: The antibodies used for Western blotting were as follows: anti-Egr-1 (sc-515830; Santa Cruz), anti-Nab2 (sc-23867; Santa Cruz), and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; GT239; GeneTex).

Techniques: Sequencing