Structured Review

Santa Cruz Biotechnology histone h3
Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated <t>histone</t> H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.
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1) Product Images from "NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor"

Article Title: NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor

Journal: International Journal of Environmental Research and Public Health

doi: 10.3390/ijerph120100064

Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.
Figure Legend Snippet: Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Isolation, Sonication, Immunoprecipitation, Amplification, Software

2) Product Images from "SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits"

Article Title: SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits

Journal: Nature Communications

doi: 10.1038/ncomms16063

Depleting SRSF1 or inhibiting its repeat-RNA sequestration inhibit the nuclear export of C9ORF72 RAN-translated transcripts. ( a , b ) Protein:RNA UV crosslinking assays using purified recombinant proteins and 32P-end-radiolabelled G4C2x5 ( a ) and C4G2x5 ( b ) repeat RNA probes. Proteins are visualized on SDS–PAGE stained with Coomassie blue (left panels) and covalently linked RNA:protein complexes by autoradiography on PhosphoImages (right panels). ( c , d ) RNA immunoprecipitation (RIP) assays. Formaldehyde was added to the medium of live N2A cells co-transfected with G4C2x15, G4C2x38 ( c ), C4G2x15 or C4G2x39 ( d ) and either FLAG control (FLAG Ctrl), FLAG-tagged SRSF1 aa11-196 wild-type (SRSF1) or SRSF1-m4 were subjected to anti-FLAG immunoprecipitation. Purified RNA was analysed by qRT–PCR following normalization to U1 snRNA levels in three biological replicate experiments (mean±s.e.m.; two-way ANOVA with Tukey’s correction for multiple comparisons; N (qRT–PCR reactions)=6). ( e ) Western blots of N2A cells co-transfected with G4C2x38 and either Ctrl or SRSF1-RNAi plasmids or with G4C2x38 and either FLAG-tagged SRSF1 aa11-196 wild-type (SRSF1) or SRSF1-m4, subjected to cellular fractionation using hypotonic lysis to yield cytoplasmic fractions. The chromatin remodelling SSRP1 factor is used to check for potential nuclear contamination. ( f ) Cytoplasmic and total G4C2-repeat sense transcript levels were normalized to U1 snRNA levels in three biological replicate experiments prior to plotting as a ratio to account for potential changes in mRNA transcription/stability (mean±s.e.m.; one-way ANOVA with Tukey’s correction for multiple comparisons; N (qRT–PCR reactions)=6). ( g ) Western blots of Drosophila expressing G4C2x36 and either Ctrl or SRSF1-RNAi, subjected to cellular fractionation using hypotonic lysis to yield cytoplasmic fractions. Histone H3 is used to check for potential nuclear contamination. ( h ) Cytoplasmic and total G4C2-repeat sense transcript levels were normalized to Tub84b levels in three biological replicate experiments prior to plotting as a ratio to account for potential changes in mRNA transcription/stability (mean±s.e.m.; paired two-tailed t -test; N (qRT–PCR reactions)=3). In contrast to cytoplasmic levels, total levels of hexanucleotide repeat transcripts were not significantly altered upon expression of SRSF1-m4 or depletion of SRSF1 in cells or flies ( Supplementary Fig. 7 ). Statistical significance of data is indicated as follows: NS: non-significant, P ≥0.05; * P
Figure Legend Snippet: Depleting SRSF1 or inhibiting its repeat-RNA sequestration inhibit the nuclear export of C9ORF72 RAN-translated transcripts. ( a , b ) Protein:RNA UV crosslinking assays using purified recombinant proteins and 32P-end-radiolabelled G4C2x5 ( a ) and C4G2x5 ( b ) repeat RNA probes. Proteins are visualized on SDS–PAGE stained with Coomassie blue (left panels) and covalently linked RNA:protein complexes by autoradiography on PhosphoImages (right panels). ( c , d ) RNA immunoprecipitation (RIP) assays. Formaldehyde was added to the medium of live N2A cells co-transfected with G4C2x15, G4C2x38 ( c ), C4G2x15 or C4G2x39 ( d ) and either FLAG control (FLAG Ctrl), FLAG-tagged SRSF1 aa11-196 wild-type (SRSF1) or SRSF1-m4 were subjected to anti-FLAG immunoprecipitation. Purified RNA was analysed by qRT–PCR following normalization to U1 snRNA levels in three biological replicate experiments (mean±s.e.m.; two-way ANOVA with Tukey’s correction for multiple comparisons; N (qRT–PCR reactions)=6). ( e ) Western blots of N2A cells co-transfected with G4C2x38 and either Ctrl or SRSF1-RNAi plasmids or with G4C2x38 and either FLAG-tagged SRSF1 aa11-196 wild-type (SRSF1) or SRSF1-m4, subjected to cellular fractionation using hypotonic lysis to yield cytoplasmic fractions. The chromatin remodelling SSRP1 factor is used to check for potential nuclear contamination. ( f ) Cytoplasmic and total G4C2-repeat sense transcript levels were normalized to U1 snRNA levels in three biological replicate experiments prior to plotting as a ratio to account for potential changes in mRNA transcription/stability (mean±s.e.m.; one-way ANOVA with Tukey’s correction for multiple comparisons; N (qRT–PCR reactions)=6). ( g ) Western blots of Drosophila expressing G4C2x36 and either Ctrl or SRSF1-RNAi, subjected to cellular fractionation using hypotonic lysis to yield cytoplasmic fractions. Histone H3 is used to check for potential nuclear contamination. ( h ) Cytoplasmic and total G4C2-repeat sense transcript levels were normalized to Tub84b levels in three biological replicate experiments prior to plotting as a ratio to account for potential changes in mRNA transcription/stability (mean±s.e.m.; paired two-tailed t -test; N (qRT–PCR reactions)=3). In contrast to cytoplasmic levels, total levels of hexanucleotide repeat transcripts were not significantly altered upon expression of SRSF1-m4 or depletion of SRSF1 in cells or flies ( Supplementary Fig. 7 ). Statistical significance of data is indicated as follows: NS: non-significant, P ≥0.05; * P

Techniques Used: Purification, Recombinant, SDS Page, Staining, Autoradiography, Immunoprecipitation, Transfection, Quantitative RT-PCR, Western Blot, Cell Fractionation, Lysis, Expressing, Two Tailed Test

3) Product Images from "Skin-specific Deletion of Stearoyl-CoA Desaturase-1 Alters Skin Lipid Composition and Protects Mice from High Fat Diet-induced Obesity *"

Article Title: Skin-specific Deletion of Stearoyl-CoA Desaturase-1 Alters Skin Lipid Composition and Protects Mice from High Fat Diet-induced Obesity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.014225

Hepatic lipogenic gene expression is not decreased in HFD-fed or fasted-refed SKO mice . A and B , hepatic lipogenic gene expression ( A ) and nuclear levels of SREBP-1, SREBP-2, and histone H 3 ( B ) were measured in Lox and SKO mice after 8 weeks of chow or
Figure Legend Snippet: Hepatic lipogenic gene expression is not decreased in HFD-fed or fasted-refed SKO mice . A and B , hepatic lipogenic gene expression ( A ) and nuclear levels of SREBP-1, SREBP-2, and histone H 3 ( B ) were measured in Lox and SKO mice after 8 weeks of chow or

Techniques Used: Expressing, Mouse Assay

4) Product Images from "Boundary Element-Associated Factor 32B Connects Chromatin Domains to the Nuclear Matrix ▿"

Article Title: Boundary Element-Associated Factor 32B Connects Chromatin Domains to the Nuclear Matrix ▿

Journal:

doi: 10.1128/MCB.00305-07

BEAF and ZW5 are associated with nuclear matrix. (A) Equal amounts of protein from nuclei (N) and nuclear matrix (NM) were analyzed by Western blotting using antibodies to BEAF, ZW5, ABD-B, and histone H3. BEAF and ZW5 are retained in the matrix, but
Figure Legend Snippet: BEAF and ZW5 are associated with nuclear matrix. (A) Equal amounts of protein from nuclei (N) and nuclear matrix (NM) were analyzed by Western blotting using antibodies to BEAF, ZW5, ABD-B, and histone H3. BEAF and ZW5 are retained in the matrix, but

Techniques Used: Western Blot

5) Product Images from "?-Keto acid metabolites of naturally-occurring organoselenium compounds as inhibitors of histone deacetylase in human prostate cancer cells"

Article Title: ?-Keto acid metabolites of naturally-occurring organoselenium compounds as inhibitors of histone deacetylase in human prostate cancer cells

Journal: Cancer prevention research (Philadelphia, Pa.)

doi: 10.1158/1940-6207.CAPR-09-0047

A , Increased acetylated-histone-H3 expression in LNCaP, LNCaPC4–2 and PC-3 cell lysates. Cells were treated with MSC (50 and 200 μM), SM (50 and 200 μM), MSP (10 and 50 μM), or KMSB (10 and 50 μM) for 5 and 24-hrs.
Figure Legend Snippet: A , Increased acetylated-histone-H3 expression in LNCaP, LNCaPC4–2 and PC-3 cell lysates. Cells were treated with MSC (50 and 200 μM), SM (50 and 200 μM), MSP (10 and 50 μM), or KMSB (10 and 50 μM) for 5 and 24-hrs.

Techniques Used: Expressing

6) Product Images from "PS-341 and Histone Deacetylase Inhibitor Synergistically Induce Apoptosis in Head and Neck Squamous Cell Carcinoma Cells"

Article Title: PS-341 and Histone Deacetylase Inhibitor Synergistically Induce Apoptosis in Head and Neck Squamous Cell Carcinoma Cells

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-10-0141

Promoting histone H3 acetylation in HNSCC cells by TSA. A and B , TSA induced hyperacetylation of histone H3 in HNSCC cells. UMSCC1 and UMSCC23 cells were treated with PS-341 alone (0.5 µM), TSA alone (300 nM), or PS-341 and TSA together for indicated
Figure Legend Snippet: Promoting histone H3 acetylation in HNSCC cells by TSA. A and B , TSA induced hyperacetylation of histone H3 in HNSCC cells. UMSCC1 and UMSCC23 cells were treated with PS-341 alone (0.5 µM), TSA alone (300 nM), or PS-341 and TSA together for indicated

Techniques Used:

7) Product Images from "PS-341 and Histone Deacetylase Inhibitor Synergistically Induce Apoptosis in Head and Neck Squamous Cell Carcinoma Cells"

Article Title: PS-341 and Histone Deacetylase Inhibitor Synergistically Induce Apoptosis in Head and Neck Squamous Cell Carcinoma Cells

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-10-0141

Promoting histone H3 acetylation in HNSCC cells by TSA. A and B , TSA induced hyperacetylation of histone H3 in HNSCC cells. UMSCC1 and UMSCC23 cells were treated with PS-341 alone (0.5 µM), TSA alone (300 nM), or PS-341 and TSA together for indicated
Figure Legend Snippet: Promoting histone H3 acetylation in HNSCC cells by TSA. A and B , TSA induced hyperacetylation of histone H3 in HNSCC cells. UMSCC1 and UMSCC23 cells were treated with PS-341 alone (0.5 µM), TSA alone (300 nM), or PS-341 and TSA together for indicated

Techniques Used:

8) Product Images from "Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts"

Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3949

TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P
Figure Legend Snippet: TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P

Techniques Used: Expressing, Cell Culture, Western Blot, Incubation, Recombinant, Positive Control, Real-time Polymerase Chain Reaction

Cooperative effects of mitogen-activated protein kinase inhibition and exogenous TGF-β on fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 ng/ml exogenous recombinant human TGF-β1, and analyzed at day 4 in culture. (A) Protein lysates were examined for expression of myofibroblast-associated protein markers α-SMA, calponin, and SM22α by western blot analysis. Histone H3 was used as a loading control. (B) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bar, 100 µ m. TGF-β, transforming growth factor-β; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; i , inhibitor.
Figure Legend Snippet: Cooperative effects of mitogen-activated protein kinase inhibition and exogenous TGF-β on fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 ng/ml exogenous recombinant human TGF-β1, and analyzed at day 4 in culture. (A) Protein lysates were examined for expression of myofibroblast-associated protein markers α-SMA, calponin, and SM22α by western blot analysis. Histone H3 was used as a loading control. (B) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bar, 100 µ m. TGF-β, transforming growth factor-β; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; i , inhibitor.

Techniques Used: Inhibition, Activation Assay, Cell Culture, Recombinant, Expressing, Western Blot, Staining, Marker

Effects of TGF-βR1 inhibition on fibroblast activation induced by ERK or JNK inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of the indicated concentration of the TGF-βR1 inhibitor RepSox until day 4 and then subjected to Western blot analysis for myofibroblast markers α-SMA and calponin. Histone H3 was used as a loading control. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 ng/ml TGF-β1 and 100 nM RepSox until day 4 and then subjected to western blot analysis for myofibroblast marker α-SMA. Histone H3 was used as a loading control. (C) Western blots were analyzed using densitometry, and relative density of α-SMA/Histone H3 was compared for each mitogen-activated protein kinase inhibitor-treated or TGF-β1-treated sample compared with its corresponding TGF-βR1 inhibitor-treated sample. Statistical significance was determined using a two-tailed Student’s t-test. * P
Figure Legend Snippet: Effects of TGF-βR1 inhibition on fibroblast activation induced by ERK or JNK inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of the indicated concentration of the TGF-βR1 inhibitor RepSox until day 4 and then subjected to Western blot analysis for myofibroblast markers α-SMA and calponin. Histone H3 was used as a loading control. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 ng/ml TGF-β1 and 100 nM RepSox until day 4 and then subjected to western blot analysis for myofibroblast marker α-SMA. Histone H3 was used as a loading control. (C) Western blots were analyzed using densitometry, and relative density of α-SMA/Histone H3 was compared for each mitogen-activated protein kinase inhibitor-treated or TGF-β1-treated sample compared with its corresponding TGF-βR1 inhibitor-treated sample. Statistical significance was determined using a two-tailed Student’s t-test. * P

Techniques Used: Inhibition, Activation Assay, Cell Culture, Concentration Assay, Western Blot, Marker, Two Tailed Test

9) Product Images from "Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3 *"

Article Title: Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.571984

XBP1 was essential for disturbed flow-induced up-regulation of HO-1. A , overexpression of XBP1u up-regulated HO-1 and Akt phosphorylation in a dose-dependent manner. HUVECs were infected with Ad- XBP1u at MOI indicated for 24 h, followed by Western blot analysis. Ad-null was included to compensate the MOI. B , overexpression of XBP1u maintained a high level of Akt1 phosphorylation and HO-1 expression. HUVECs were infected with Ad- XBP1u at 10 MOI for 24 h and 48 h, followed by Western blot analysis. Ad-null was included as control. FLAG indicates the exogenous XBP1u. C , knockdown of XBP1 via shRNA lentivirus ( XBP1sh ) decreased basal level of Akt1 phosphorylation and HO-1 expression. Non-target shRNA lentivirus ( NTsh ) was included as control. D , knockdown of XBP1 via shRNA lentivirus ( XBP1sh ) abolished disturbed flow-induced HO-1 expression. Non-target shRNA lentivirus ( NTsh ) was included as control. E , Nrf2 was necessary for flow-induced HO-1 expression. HUVECs were transfected with control siRNA ( CTLsi ) or Nrf2 siRNA ( Nrf2si ) for 72 h, followed by disturbed flow for 4 h. F , flow stabilized Nrf2 via post-translational modification. HUVECs were treated with 1 μmol/liter actinomycin D ( AD ) or 30 mg/liter cycloheximide ( CH ) for 1 h, followed by disturbed flow for 4 h or kept at static conditions in the presence of the inhibitors. DMSO ( DM ) was included as vehicle control. G , AZD2014 abolished Ad-XBP1u ( X1u ) or Ad-HDAC3 ( HD3 )-induced pAkt Ser-473 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. HUVECs were infected with Ad-null or Ad- XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by cellular fraction isolation and Western blot analysis. DMSO was included as vehicle control. The anti-FLAG antibody was included to detect exogenous XBP1u and HDAC3. Antibodies against α-tubulin and histone H3 were included to indicate cytosol and nuclear extract, respectively. The samples from cytosol and nuclear extraction were run on separate gels but performed Western blot at the same time and exposed to x-ray film exactly at the same time period. H , AZD2014 attenuated XBP1u/HDAC3-induced Akt1 phosphorylation in nucleus. HUVECs were infected with Ad-null or Ad- XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by double immunofluorescence staining with anti-mTOR ( red ) and anti-pAkt Ser-473 ( green ) antibodies. I , AZD2014 reduced flow-induced Nrf2 nuclear translocation. HUVECs were treated with 5 μmol/liter AZD2014 for 1 h, followed by disturbed flow for 4 h or being kept at static conditions in the presence of ZAD2014. DMSO was included as vehicle control. Double immunofluorescence staining was performed with anti-mTOR ( red ) and anti-Nrf2 ( green ) or pAkt Ser-473 ( green ) antibodies. Data presented are representatives of three independent experiments.
Figure Legend Snippet: XBP1 was essential for disturbed flow-induced up-regulation of HO-1. A , overexpression of XBP1u up-regulated HO-1 and Akt phosphorylation in a dose-dependent manner. HUVECs were infected with Ad- XBP1u at MOI indicated for 24 h, followed by Western blot analysis. Ad-null was included to compensate the MOI. B , overexpression of XBP1u maintained a high level of Akt1 phosphorylation and HO-1 expression. HUVECs were infected with Ad- XBP1u at 10 MOI for 24 h and 48 h, followed by Western blot analysis. Ad-null was included as control. FLAG indicates the exogenous XBP1u. C , knockdown of XBP1 via shRNA lentivirus ( XBP1sh ) decreased basal level of Akt1 phosphorylation and HO-1 expression. Non-target shRNA lentivirus ( NTsh ) was included as control. D , knockdown of XBP1 via shRNA lentivirus ( XBP1sh ) abolished disturbed flow-induced HO-1 expression. Non-target shRNA lentivirus ( NTsh ) was included as control. E , Nrf2 was necessary for flow-induced HO-1 expression. HUVECs were transfected with control siRNA ( CTLsi ) or Nrf2 siRNA ( Nrf2si ) for 72 h, followed by disturbed flow for 4 h. F , flow stabilized Nrf2 via post-translational modification. HUVECs were treated with 1 μmol/liter actinomycin D ( AD ) or 30 mg/liter cycloheximide ( CH ) for 1 h, followed by disturbed flow for 4 h or kept at static conditions in the presence of the inhibitors. DMSO ( DM ) was included as vehicle control. G , AZD2014 abolished Ad-XBP1u ( X1u ) or Ad-HDAC3 ( HD3 )-induced pAkt Ser-473 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. HUVECs were infected with Ad-null or Ad- XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by cellular fraction isolation and Western blot analysis. DMSO was included as vehicle control. The anti-FLAG antibody was included to detect exogenous XBP1u and HDAC3. Antibodies against α-tubulin and histone H3 were included to indicate cytosol and nuclear extract, respectively. The samples from cytosol and nuclear extraction were run on separate gels but performed Western blot at the same time and exposed to x-ray film exactly at the same time period. H , AZD2014 attenuated XBP1u/HDAC3-induced Akt1 phosphorylation in nucleus. HUVECs were infected with Ad-null or Ad- XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by double immunofluorescence staining with anti-mTOR ( red ) and anti-pAkt Ser-473 ( green ) antibodies. I , AZD2014 reduced flow-induced Nrf2 nuclear translocation. HUVECs were treated with 5 μmol/liter AZD2014 for 1 h, followed by disturbed flow for 4 h or being kept at static conditions in the presence of ZAD2014. DMSO was included as vehicle control. Double immunofluorescence staining was performed with anti-mTOR ( red ) and anti-Nrf2 ( green ) or pAkt Ser-473 ( green ) antibodies. Data presented are representatives of three independent experiments.

Techniques Used: Flow Cytometry, Over Expression, Infection, Western Blot, Expressing, shRNA, Transfection, Modification, Translocation Assay, Isolation, Double Immunofluorescence Staining

10) Product Images from "Regulation of XPC deubiquitination by USP11 in repair of UV-induced DNA damage"

Article Title: Regulation of XPC deubiquitination by USP11 in repair of UV-induced DNA damage

Journal: Oncotarget

doi: 10.18632/oncotarget.22105

UVB induces USP11 recruitment to the chromatin ( A, B ) Immunoblot analysis of USP11 and GAPDH in HaCaT cells (A), and normal human epidermal keratinocyte (NHEK) cells (B) with or without MG132 treatment (10 µM) 1 h prior to UVB (20 mJ/cm 2 ). ( C ) Immunoblot analysis of USP11, GAPDH, and Lamin B using nuclear [N] and cytoplasmic [C] fractions from HaCaT cells with or without UVB (20 mJ/cm 2 , 30 min). ( D ) Immunoblot analysis of USP11, Histone 3, and XPC using chromatin bound protein fractions from HaCaT cells over a time course post-UVB (20 mJ/cm 2 ).
Figure Legend Snippet: UVB induces USP11 recruitment to the chromatin ( A, B ) Immunoblot analysis of USP11 and GAPDH in HaCaT cells (A), and normal human epidermal keratinocyte (NHEK) cells (B) with or without MG132 treatment (10 µM) 1 h prior to UVB (20 mJ/cm 2 ). ( C ) Immunoblot analysis of USP11, GAPDH, and Lamin B using nuclear [N] and cytoplasmic [C] fractions from HaCaT cells with or without UVB (20 mJ/cm 2 , 30 min). ( D ) Immunoblot analysis of USP11, Histone 3, and XPC using chromatin bound protein fractions from HaCaT cells over a time course post-UVB (20 mJ/cm 2 ).

Techniques Used:

USP11 deubiquitinates XPC at the chromatin following UVB damage ( A, B ) Immunoblot analysis of XPC, USP11, and GAPDH in HaCaT cells transfected with siUSP11 or siNC and treated with or without MG132 (10 µM) 1 h prior to UVB exposure (20 mJ/cm 2 ) (A), 293T WT and ΔUSP11 cells (B) at the indicated times post-UVB (20 mJ/cm 2 ). ( C ) Immunoblot analysis of XPC and histone H3 using chromatin-bound protein fractions from HaCaT cells stably infected with a lentiviral vector expressing shCon or shUSP11 at the indicated times post-sham or -UVB (20 mJ/cm 2 ) irradiation.
Figure Legend Snippet: USP11 deubiquitinates XPC at the chromatin following UVB damage ( A, B ) Immunoblot analysis of XPC, USP11, and GAPDH in HaCaT cells transfected with siUSP11 or siNC and treated with or without MG132 (10 µM) 1 h prior to UVB exposure (20 mJ/cm 2 ) (A), 293T WT and ΔUSP11 cells (B) at the indicated times post-UVB (20 mJ/cm 2 ). ( C ) Immunoblot analysis of XPC and histone H3 using chromatin-bound protein fractions from HaCaT cells stably infected with a lentiviral vector expressing shCon or shUSP11 at the indicated times post-sham or -UVB (20 mJ/cm 2 ) irradiation.

Techniques Used: Transfection, Stable Transfection, Infection, Plasmid Preparation, Expressing, Irradiation

11) Product Images from "Vorinostat enhances the anticancer effect of oxaliplatin on hepatocellular carcinoma cells"

Article Title: Vorinostat enhances the anticancer effect of oxaliplatin on hepatocellular carcinoma cells

Journal: Cancer Medicine

doi: 10.1002/cam4.1278

Vorinostat and oxaliplatin attenuated the proliferation of HCC cell lines. (A) Cytotoxicity assay. HepG2, SMMC 7721, and BEL 7402 cells in 96‐well plates were treated with different concentrations of vorinostat and oxaliplatin for 48 h and cell viability was detected by the Cell Counting Kit‐8 ( CCK ‐8) assay. The half maximal inhibitory concentration ( IC 50) was calculated by SPSS software. (B) HepG2 and SMMC 7721 cells were treated with 1 μ mol/L vorinostat and BEL 7402 cells were treated with 5 μ mol/L vorinostat. The expression of acetylated Histone H3 was detected by western blotting. (C) Combination index ( CI ) values of HepG2, SMMC 7721, and BEL 7402 cells at different fractions affected. (D) Dose‐reduction index ( DRI ) values of HepG2, SMMC 7721, and BEL 7402 cells at different fractions affected. All experiments were performed at least three independent times.
Figure Legend Snippet: Vorinostat and oxaliplatin attenuated the proliferation of HCC cell lines. (A) Cytotoxicity assay. HepG2, SMMC 7721, and BEL 7402 cells in 96‐well plates were treated with different concentrations of vorinostat and oxaliplatin for 48 h and cell viability was detected by the Cell Counting Kit‐8 ( CCK ‐8) assay. The half maximal inhibitory concentration ( IC 50) was calculated by SPSS software. (B) HepG2 and SMMC 7721 cells were treated with 1 μ mol/L vorinostat and BEL 7402 cells were treated with 5 μ mol/L vorinostat. The expression of acetylated Histone H3 was detected by western blotting. (C) Combination index ( CI ) values of HepG2, SMMC 7721, and BEL 7402 cells at different fractions affected. (D) Dose‐reduction index ( DRI ) values of HepG2, SMMC 7721, and BEL 7402 cells at different fractions affected. All experiments were performed at least three independent times.

Techniques Used: Cytotoxicity Assay, Cell Counting, CCK-8 Assay, Concentration Assay, Software, Expressing, Western Blot

12) Product Images from "Inflamed macrophage microvesicles induce insulin resistance in human adipocytes"

Article Title: Inflamed macrophage microvesicles induce insulin resistance in human adipocytes

Journal: Nutrition & Metabolism

doi: 10.1186/s12986-015-0016-3

Effect of macrophage-derived MVs on insulin-stimulated activation of NF-κB in human adipocytes. a , b . Relative protein levels of nuclear NF-κB p65 in human mature adipocytes ( a ) and differentiated cells ( b ) were analyzed by western blotting. Human adipocytes were treated with indicated MVs for 24 h, and then incubated with 0, 1, 10, or 100 nM of insulin for 20 min. NF-κB p65 and histone H3 levels in nuclear lysates were determined by immunoblotting. Quantification of NF-κB p65 protein level is shown below. Western blot images were analyzed using Bandscan software, and statistical analysis is presented below. * P
Figure Legend Snippet: Effect of macrophage-derived MVs on insulin-stimulated activation of NF-κB in human adipocytes. a , b . Relative protein levels of nuclear NF-κB p65 in human mature adipocytes ( a ) and differentiated cells ( b ) were analyzed by western blotting. Human adipocytes were treated with indicated MVs for 24 h, and then incubated with 0, 1, 10, or 100 nM of insulin for 20 min. NF-κB p65 and histone H3 levels in nuclear lysates were determined by immunoblotting. Quantification of NF-κB p65 protein level is shown below. Western blot images were analyzed using Bandscan software, and statistical analysis is presented below. * P

Techniques Used: Derivative Assay, Activation Assay, Western Blot, Incubation, Software

Blocking of NF-κB reverse the inhibitory effect of M1 MVs on pAkt level and GLUT4 translocation in human adipocytes. a , d . Expression level of nuclear NF-κB p65 in human mature adipocytes ( a ) and differentiated cells ( d ) were analyzed by western blotting. Adipocytes were pretreated with or without NF-κB specific inhibitor (Bay, 10 μM) for 2 h, then incubated with M1 MVs for 24 h. Nuclear lysates were analyzed for NF-κB p65 and histone H3 level after insulin stimulation for a further 20 min. Quantification of NF-κB p65 protein level is presented below. b , e . Protein level of pAkt in human mature adipocytes ( b ) and differentiated adipocytes ( e ), after treatment as in a . Western blot images were analyzed using Bandscan software, and statistical analysis is presented below. c , f . The levels of GLUT4 in plasma membrane (PM) fractions of human mature adipocytes ( c ) and differentiated adipocytes ( f ) were determined by western blotting. Cells were treated as in A, and PM fractions were obtained and subjected to immunoblotting with the indicated antibodies. Quantification of the GLUT4 protein level is shown below. * P
Figure Legend Snippet: Blocking of NF-κB reverse the inhibitory effect of M1 MVs on pAkt level and GLUT4 translocation in human adipocytes. a , d . Expression level of nuclear NF-κB p65 in human mature adipocytes ( a ) and differentiated cells ( d ) were analyzed by western blotting. Adipocytes were pretreated with or without NF-κB specific inhibitor (Bay, 10 μM) for 2 h, then incubated with M1 MVs for 24 h. Nuclear lysates were analyzed for NF-κB p65 and histone H3 level after insulin stimulation for a further 20 min. Quantification of NF-κB p65 protein level is presented below. b , e . Protein level of pAkt in human mature adipocytes ( b ) and differentiated adipocytes ( e ), after treatment as in a . Western blot images were analyzed using Bandscan software, and statistical analysis is presented below. c , f . The levels of GLUT4 in plasma membrane (PM) fractions of human mature adipocytes ( c ) and differentiated adipocytes ( f ) were determined by western blotting. Cells were treated as in A, and PM fractions were obtained and subjected to immunoblotting with the indicated antibodies. Quantification of the GLUT4 protein level is shown below. * P

Techniques Used: Blocking Assay, Translocation Assay, Expressing, Western Blot, Incubation, Software

13) Product Images from "Low and high concentrations of butyrate regulate fat accumulation in chicken adipocytes via different mechanisms"

Article Title: Low and high concentrations of butyrate regulate fat accumulation in chicken adipocytes via different mechanisms

Journal: Adipocyte

doi: 10.1080/21623945.2020.1738791

Signalling elucidation of the role of 1 mM SB in fat accumulation. (a)Effect of 1 mM SB on phosphorylation of ERK and AMPK at the indicated time points, determined by western blotting and quantification analysis. GAPDH serves as a loading control. This experiment was repeated 3 times. (b) HDAC activity in the treated cells, determined using a commercial assay kit. (c) Oil red O staining (red) was performed in differentiated adipocytes treated with SB or TSA for 8 days. The nuclei were stained with haematoxylin (purple). (d) Quantification of the accumulated TG based on the same protein content. (e) Relative mRNA expression levels of PPARG, FAS, APN, LPL and FABP4 in treated cells. (f) Relative mRNA expression levels of FFAR2 and FFAR3 in treated cells. (g) Representative images of western blots and quantitative analysis of PPARG, FABP4, histone H3 and acetyl-histone H3 in the treated cells (n = 3). GAPDH was used as an internal control. The data are the means ± SEM (n = 3_6). * p
Figure Legend Snippet: Signalling elucidation of the role of 1 mM SB in fat accumulation. (a)Effect of 1 mM SB on phosphorylation of ERK and AMPK at the indicated time points, determined by western blotting and quantification analysis. GAPDH serves as a loading control. This experiment was repeated 3 times. (b) HDAC activity in the treated cells, determined using a commercial assay kit. (c) Oil red O staining (red) was performed in differentiated adipocytes treated with SB or TSA for 8 days. The nuclei were stained with haematoxylin (purple). (d) Quantification of the accumulated TG based on the same protein content. (e) Relative mRNA expression levels of PPARG, FAS, APN, LPL and FABP4 in treated cells. (f) Relative mRNA expression levels of FFAR2 and FFAR3 in treated cells. (g) Representative images of western blots and quantitative analysis of PPARG, FABP4, histone H3 and acetyl-histone H3 in the treated cells (n = 3). GAPDH was used as an internal control. The data are the means ± SEM (n = 3_6). * p

Techniques Used: Western Blot, Activity Assay, Staining, Expressing

14) Product Images from "Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy"

Article Title: Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0201858

BZ increases nuclear p65 accumulation, and IKK-dependent p65 recruitment to IL-8 promoter in TNBC cells. (A) Western blotting of cytoplasmic (CE) and nuclear extracts (NE) prepared from MDA-MB-231 cells treated 24 h with BZ, and analyzed by using p65 and IκBα antibodies. The presence of cytoplasmic proteins in nuclear fraction was evaluated by re-probing the membrane with lactate dehydrogenase (LDH) antibody. Nuclear contamination in the cytoplasmic fraction was assessed using histone H3 specific antibody. To confirm equal protein loading, the membranes were stripped and re-probed with actin antibody. Each lane corresponds to approximately 5×10 4 cells. (B) Densitometric evaluation of p65, IκBα, and histone H3 levels in nuclear extracts of BZ-treated MDA-MB-231 cells, shown in panel A. The densities of nuclear p65, IκBα, and histone H3 were normalized to the densities of nuclear actin. The values for untreated cells were arbitrarily set to 1, and the other values are presented relative to these values. (C) Recruitment of p65 to endogenous IL-8 promoter analyzed by ChIP and quantified by real time PCR in MDA-MB-231 cells treated with 100 nM BZ for 0, 3, 6 and 24 h. (D) ChIP of p65 recruitment to IL-8 promoter in MDA-MB-231 cells pre-incubated 12 h with control DMSO, Bay-117082 (10 μM), or SC-514 (10 μM), and then incubated 24 h with 100 BZ. The data are presented as the change in occupancy over the human IGX1A (Qiagen) sequence control and represent the mean +/− SE of three experiments. Asterisks denote a statistically significant change compared to control.
Figure Legend Snippet: BZ increases nuclear p65 accumulation, and IKK-dependent p65 recruitment to IL-8 promoter in TNBC cells. (A) Western blotting of cytoplasmic (CE) and nuclear extracts (NE) prepared from MDA-MB-231 cells treated 24 h with BZ, and analyzed by using p65 and IκBα antibodies. The presence of cytoplasmic proteins in nuclear fraction was evaluated by re-probing the membrane with lactate dehydrogenase (LDH) antibody. Nuclear contamination in the cytoplasmic fraction was assessed using histone H3 specific antibody. To confirm equal protein loading, the membranes were stripped and re-probed with actin antibody. Each lane corresponds to approximately 5×10 4 cells. (B) Densitometric evaluation of p65, IκBα, and histone H3 levels in nuclear extracts of BZ-treated MDA-MB-231 cells, shown in panel A. The densities of nuclear p65, IκBα, and histone H3 were normalized to the densities of nuclear actin. The values for untreated cells were arbitrarily set to 1, and the other values are presented relative to these values. (C) Recruitment of p65 to endogenous IL-8 promoter analyzed by ChIP and quantified by real time PCR in MDA-MB-231 cells treated with 100 nM BZ for 0, 3, 6 and 24 h. (D) ChIP of p65 recruitment to IL-8 promoter in MDA-MB-231 cells pre-incubated 12 h with control DMSO, Bay-117082 (10 μM), or SC-514 (10 μM), and then incubated 24 h with 100 BZ. The data are presented as the change in occupancy over the human IGX1A (Qiagen) sequence control and represent the mean +/− SE of three experiments. Asterisks denote a statistically significant change compared to control.

Techniques Used: Western Blot, Multiple Displacement Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Incubation, Sequencing

15) Product Images from "NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor"

Article Title: NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor

Journal: International Journal of Environmental Research and Public Health

doi: 10.3390/ijerph120100064

Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.
Figure Legend Snippet: Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Isolation, Sonication, Immunoprecipitation, Amplification, Software

16) Product Images from "Taeumjowi-tang, a Traditional Korean Sasang Remedy, Improves Obesity-Atopic Dermatitis Comorbidity by Regulating Hypoxia-Inducible Factor 1 Alpha"

Article Title: Taeumjowi-tang, a Traditional Korean Sasang Remedy, Improves Obesity-Atopic Dermatitis Comorbidity by Regulating Hypoxia-Inducible Factor 1 Alpha

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.01458

TJT improves obesity-AD comorbidity  via  HIF-1α regulation.  (A)  A western blot assay was performed to examine HIF-1α expression in an obesity-AD comorbidity vitro model of LPS-activated, adipocyte CM-treated HaCaT cells.  (B)  Localization of NF-κB is shown by IF staining of adipocyte CM-treated HaCaT cells.  (C)  Expression levels of HIF-1α, cytosolic and nuclear NF-κB and p-IκB were determined by western blot assays. The data are represented as the mean ± SEM of three or more experiments. HIF-1α was normalized to GAPDH, p-IκB was normalized to t-IκB, and nuclear NF-κB was normalized to histone H3. * P
Figure Legend Snippet: TJT improves obesity-AD comorbidity via HIF-1α regulation. (A) A western blot assay was performed to examine HIF-1α expression in an obesity-AD comorbidity vitro model of LPS-activated, adipocyte CM-treated HaCaT cells. (B) Localization of NF-κB is shown by IF staining of adipocyte CM-treated HaCaT cells. (C) Expression levels of HIF-1α, cytosolic and nuclear NF-κB and p-IκB were determined by western blot assays. The data are represented as the mean ± SEM of three or more experiments. HIF-1α was normalized to GAPDH, p-IκB was normalized to t-IκB, and nuclear NF-κB was normalized to histone H3. * P

Techniques Used: Western Blot, Expressing, Staining

17) Product Images from "Long Non Coding RNA H19: A New Player in Hypoxia-Induced Multiple Myeloma Cell Dissemination"

Article Title: Long Non Coding RNA H19: A New Player in Hypoxia-Induced Multiple Myeloma Cell Dissemination

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20040801

qRT-PCR ( A ) and Western blot analysis on total protein extract ( B ) of HIF-1α expression in MM cells silenced or not for lncH19. Densitometric analysis with Image J software was done with respect to total protein level of β-actin, used as loading control. ( C ) Western blot analysis on nuclear extract of MM hypoxic cells silenced or not for lncH19. Densitometric analysis with Image J software was done with respect to nuclear protein level of histone H3, used as loading control. Values are presented as mean ± SD.
Figure Legend Snippet: qRT-PCR ( A ) and Western blot analysis on total protein extract ( B ) of HIF-1α expression in MM cells silenced or not for lncH19. Densitometric analysis with Image J software was done with respect to total protein level of β-actin, used as loading control. ( C ) Western blot analysis on nuclear extract of MM hypoxic cells silenced or not for lncH19. Densitometric analysis with Image J software was done with respect to nuclear protein level of histone H3, used as loading control. Values are presented as mean ± SD.

Techniques Used: Quantitative RT-PCR, Western Blot, Expressing, Software

18) Product Images from "Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts"

Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3949

TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P
Figure Legend Snippet: TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P

Techniques Used: Expressing, Cell Culture, Western Blot, Incubation, Recombinant, Positive Control, Real-time Polymerase Chain Reaction

Effects of TGF-βR1 inhibition on fibroblast activation induced by ERK or JNK inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of the indicated concentration of the TGF-βR1 inhibitor RepSox until day 4 and then subjected to Western blot analysis for myofibroblast markers α-SMA and calponin. Histone H3 was used as a loading control. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 ng/ml TGF-β1 and 100 nM RepSox until day 4 and then subjected to western blot analysis for myofibroblast marker α-SMA. Histone H3 was used as a loading control. (C) Western blots were analyzed using densitometry, and relative density of α-SMA/Histone H3 was compared for each mitogen-activated protein kinase inhibitor-treated or TGF-β1-treated sample compared with its corresponding TGF-βR1 inhibitor-treated sample. Statistical significance was determined using a two-tailed Student’s t-test. * P
Figure Legend Snippet: Effects of TGF-βR1 inhibition on fibroblast activation induced by ERK or JNK inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of the indicated concentration of the TGF-βR1 inhibitor RepSox until day 4 and then subjected to Western blot analysis for myofibroblast markers α-SMA and calponin. Histone H3 was used as a loading control. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 ng/ml TGF-β1 and 100 nM RepSox until day 4 and then subjected to western blot analysis for myofibroblast marker α-SMA. Histone H3 was used as a loading control. (C) Western blots were analyzed using densitometry, and relative density of α-SMA/Histone H3 was compared for each mitogen-activated protein kinase inhibitor-treated or TGF-β1-treated sample compared with its corresponding TGF-βR1 inhibitor-treated sample. Statistical significance was determined using a two-tailed Student’s t-test. * P

Techniques Used: Inhibition, Activation Assay, Cell Culture, Concentration Assay, Western Blot, Marker, Two Tailed Test

Cooperative effects of mitogen-activated protein kinase inhibition and exogenous TGF-β on fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 ng/ml exogenous recombinant human TGF-β1, and analyzed at day 4 in culture. (A) Protein lysates were examined for expression of myofibroblast-associated protein markers α-SMA, calponin, and SM22α by western blot analysis. Histone H3 was used as a loading control. (B) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bar, 100 µ m. TGF-β, transforming growth factor-β; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; i , inhibitor.
Figure Legend Snippet: Cooperative effects of mitogen-activated protein kinase inhibition and exogenous TGF-β on fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 ng/ml exogenous recombinant human TGF-β1, and analyzed at day 4 in culture. (A) Protein lysates were examined for expression of myofibroblast-associated protein markers α-SMA, calponin, and SM22α by western blot analysis. Histone H3 was used as a loading control. (B) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bar, 100 µ m. TGF-β, transforming growth factor-β; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; i , inhibitor.

Techniques Used: Inhibition, Activation Assay, Cell Culture, Recombinant, Expressing, Western Blot, Staining, Marker

19) Product Images from "Molecular Mechanism of the Cell Death Induced by the Histone Deacetylase Pan Inhibitor LBH589 (Panobinostat) in Wilms Tumor Cells"

Article Title: Molecular Mechanism of the Cell Death Induced by the Histone Deacetylase Pan Inhibitor LBH589 (Panobinostat) in Wilms Tumor Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0126566

Real-time PCR and western blot analysis validation of LBH589-regulated genes in LBH589-treated SK-NEP-1 cells. (A) Quantitative RT-PCR analysis of RPRM , DHRS2 , DNAJA3 , STMN2 , PRKACA , PAM , PTPN7 and EIF2AK2 in LBH589-treated SK-NEP-1 cells. (B) Western blot analysis of AC Histone H3K9, AC Histone H4K8, Histone H3, c-MYC, PRKCA, RPRM and DNAJA3 in LBH589-treated SK-NEP-1 cells.
Figure Legend Snippet: Real-time PCR and western blot analysis validation of LBH589-regulated genes in LBH589-treated SK-NEP-1 cells. (A) Quantitative RT-PCR analysis of RPRM , DHRS2 , DNAJA3 , STMN2 , PRKACA , PAM , PTPN7 and EIF2AK2 in LBH589-treated SK-NEP-1 cells. (B) Western blot analysis of AC Histone H3K9, AC Histone H4K8, Histone H3, c-MYC, PRKCA, RPRM and DNAJA3 in LBH589-treated SK-NEP-1 cells.

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR

20) Product Images from "Sulforaphane prevents doxorubicin-induced oxidative stress and cell death in rat H9c2 cells"

Article Title: Sulforaphane prevents doxorubicin-induced oxidative stress and cell death in rat H9c2 cells

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2015.2199

Activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2) pathway by L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) in H9c2 cells. (A) H9c2 cells were treated with 1 µ M doxorubicin (Dox) or pre-treated with 10 µ M L-Sul or D,L-Sul for 2 h, and then treated with 1 µ M Dox for 24 h. Cells were double immunostained for Nrf2 and Keap1 and nuclei were visualized by DAPI staining. (B) H9c2 cells were treated with 1 µ M Dox or pre-treated with 10 µ M L-Sul or D,L-Sul for 2 h, and then treated with 1 µ M Dox for 24 h. In parallel, cells were also treated with 10 µ M L-Sul or D,L-Sul alone for 24 h. Nuclear and cytosolic fractions of H9c2 cells were obtained and subjected to western blot analysis using Nrf2 and Keap1 antibodies (top panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a cytosolic marker, while histone H3 was used to identify nuclear fractions. Densitometric analysis is shown on the lower panel. # P
Figure Legend Snippet: Activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2) pathway by L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) in H9c2 cells. (A) H9c2 cells were treated with 1 µ M doxorubicin (Dox) or pre-treated with 10 µ M L-Sul or D,L-Sul for 2 h, and then treated with 1 µ M Dox for 24 h. Cells were double immunostained for Nrf2 and Keap1 and nuclei were visualized by DAPI staining. (B) H9c2 cells were treated with 1 µ M Dox or pre-treated with 10 µ M L-Sul or D,L-Sul for 2 h, and then treated with 1 µ M Dox for 24 h. In parallel, cells were also treated with 10 µ M L-Sul or D,L-Sul alone for 24 h. Nuclear and cytosolic fractions of H9c2 cells were obtained and subjected to western blot analysis using Nrf2 and Keap1 antibodies (top panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a cytosolic marker, while histone H3 was used to identify nuclear fractions. Densitometric analysis is shown on the lower panel. # P

Techniques Used: Activation Assay, Staining, Western Blot, Marker

21) Product Images from "Panhistone deacetylase inhibitors inhibit proinflammatory signaling pathways to ameliorate interleukin-18-induced cardiac hypertrophy"

Article Title: Panhistone deacetylase inhibitors inhibit proinflammatory signaling pathways to ameliorate interleukin-18-induced cardiac hypertrophy

Journal: Physiological Genomics

doi: 10.1152/physiolgenomics.00048.2011

Posttranslational modifications of histones in the cardiac chromatin of mice treated with IL-18 ± TSA or CBHA. Mice were injected with IL-18 with or without CBHA or TSA. We separated 3 μg aliquots of protein extracts from individual mouse heart by 15% SDS-PAGE. Western blots were sequentially probed with mono-specific antibodies against histone H3-K9ac, H3-K9me3, or H3-S10-PO 4 , or unmodified histone H3. A : acetylation of histone H3 (H3-K9) in mice treated with IL-18 ± CBHA or TSA. A ratio of acetylated H3-K9 to total histone H3 in cardiac chromatin of mice representing different treatment cohorts was calculated by densitometry. Each point represents data from an individual mouse heart. Values are shown as means (horizontal bar) ± SD. P values of TSA, CBHA, TSA + IL-18, CBHA + IL-18 vs. CON are P
Figure Legend Snippet: Posttranslational modifications of histones in the cardiac chromatin of mice treated with IL-18 ± TSA or CBHA. Mice were injected with IL-18 with or without CBHA or TSA. We separated 3 μg aliquots of protein extracts from individual mouse heart by 15% SDS-PAGE. Western blots were sequentially probed with mono-specific antibodies against histone H3-K9ac, H3-K9me3, or H3-S10-PO 4 , or unmodified histone H3. A : acetylation of histone H3 (H3-K9) in mice treated with IL-18 ± CBHA or TSA. A ratio of acetylated H3-K9 to total histone H3 in cardiac chromatin of mice representing different treatment cohorts was calculated by densitometry. Each point represents data from an individual mouse heart. Values are shown as means (horizontal bar) ± SD. P values of TSA, CBHA, TSA + IL-18, CBHA + IL-18 vs. CON are P

Techniques Used: Mouse Assay, Injection, SDS Page, Western Blot

22) Product Images from "Prevention of Wogonin on Colorectal Cancer Tumorigenesis by Regulating p53 Nuclear Translocation"

Article Title: Prevention of Wogonin on Colorectal Cancer Tumorigenesis by Regulating p53 Nuclear Translocation

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.01356

Wogonin modulated nuclear translocation of p-p53 through increased ER stress. (A) Total protein, cytoplasm protein and nuclear protein expressions of p53(ser315) andp53 (ser376) were examined by western blot after wogonin treated for 72 h in HCT-116 cells. Histone H3 or GAPDH was used as an internal control. (B) The distribution of p53(ser15) were visualized by immunofluorescence using p53(ser15) antibody (Green), nuclei were counterstained with DAPI (Blue) and cytomembrane were counterstained with phalloidine (Red). p53(ser15) fluorescence intensity ratio of cytoplasm/nucleus was expressed in histogram. (C) Immunofluorescence assay was conducted to evaluate the ratio of phosphorylation at serine 15 of p53 in nucleus/cytoplasm. HCT116 cells were treated with wogonin (10 μM), TUDCA (500 μM), and wogonin combined with TUDCA for 48 h in HCT-116 cells. Values represent mean ± SD, significant difference versus control group, ∗∗∗ P
Figure Legend Snippet: Wogonin modulated nuclear translocation of p-p53 through increased ER stress. (A) Total protein, cytoplasm protein and nuclear protein expressions of p53(ser315) andp53 (ser376) were examined by western blot after wogonin treated for 72 h in HCT-116 cells. Histone H3 or GAPDH was used as an internal control. (B) The distribution of p53(ser15) were visualized by immunofluorescence using p53(ser15) antibody (Green), nuclei were counterstained with DAPI (Blue) and cytomembrane were counterstained with phalloidine (Red). p53(ser15) fluorescence intensity ratio of cytoplasm/nucleus was expressed in histogram. (C) Immunofluorescence assay was conducted to evaluate the ratio of phosphorylation at serine 15 of p53 in nucleus/cytoplasm. HCT116 cells were treated with wogonin (10 μM), TUDCA (500 μM), and wogonin combined with TUDCA for 48 h in HCT-116 cells. Values represent mean ± SD, significant difference versus control group, ∗∗∗ P

Techniques Used: Translocation Assay, Western Blot, Immunofluorescence, Fluorescence

23) Product Images from "K12-biotinylated histone H4 is enriched in telomeric repeats from human lung IMR-90 fibroblasts"

Article Title: K12-biotinylated histone H4 is enriched in telomeric repeats from human lung IMR-90 fibroblasts

Journal: The Journal of nutritional biochemistry

doi: 10.1016/j.jnutbio.2009.01.010

H4K12bio decreases in senescent IMR-90 fibroblasts but not in hTERT immortalized fibroblasts. Bulk histones were extracted from cells at different population doublings (PD) and biotin was probed as follows. (A) Streptavidin; (B) anti-H4K12bio (C) anti-H3K9bio;
Figure Legend Snippet: H4K12bio decreases in senescent IMR-90 fibroblasts but not in hTERT immortalized fibroblasts. Bulk histones were extracted from cells at different population doublings (PD) and biotin was probed as follows. (A) Streptavidin; (B) anti-H4K12bio (C) anti-H3K9bio;

Techniques Used:

H4K12bio is associated with telomeric repeats. Chromatin was immunoprecipitated from hTERT and IMR-90 cells using antibodies to H4K12bio, the C-terminus in histone H3, TRF2, and preimmune serum (negative control); DNA was amplified by ligation-mediated
Figure Legend Snippet: H4K12bio is associated with telomeric repeats. Chromatin was immunoprecipitated from hTERT and IMR-90 cells using antibodies to H4K12bio, the C-terminus in histone H3, TRF2, and preimmune serum (negative control); DNA was amplified by ligation-mediated

Techniques Used: Immunoprecipitation, Negative Control, Amplification, Ligation

24) Product Images from "Apigenin Alleviates Endotoxin-Induced Myocardial Toxicity by Modulating Inflammation, Oxidative Stress, and Autophagy"

Article Title: Apigenin Alleviates Endotoxin-Induced Myocardial Toxicity by Modulating Inflammation, Oxidative Stress, and Autophagy

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2017/2302896

Apigenin enhances LPS-induced TFEB nuclear localization and its downstream genes Vps11 and Map1lc3. (a) Western blot analyses of TFEB in nuclear and cytoplasmic fraction from heart lysates. Histone H3 was used as a nuclear control whereas tubulin was used as a cytoplasmic control. (b) TFEB-regulated genes Map1lc3 and Vps11 were examined at mRNA level by real-time PCR. LPS induced mRNA level in both genes whereas apigenin further enhanced those mRNA expression. Values represented as means ± SD; ∗ P
Figure Legend Snippet: Apigenin enhances LPS-induced TFEB nuclear localization and its downstream genes Vps11 and Map1lc3. (a) Western blot analyses of TFEB in nuclear and cytoplasmic fraction from heart lysates. Histone H3 was used as a nuclear control whereas tubulin was used as a cytoplasmic control. (b) TFEB-regulated genes Map1lc3 and Vps11 were examined at mRNA level by real-time PCR. LPS induced mRNA level in both genes whereas apigenin further enhanced those mRNA expression. Values represented as means ± SD; ∗ P

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing

Apigenin attenuates LPS-induced cardiac NF κ B nuclear translocation and oxidative damage. (a) Western blot analyses of NF κ B (p65) in nuclear and cytoplasmic fractions from heart lysates. Histone H3 was used as a nuclear control whereas tubulin was used as a cytoplasmic control. (b) Cardiac oxidative markers protein nitration and carbonyl content measured by quantitative ELISA. All markers were significantly increased in LPS-treated mice. Apigenin significantly reduced LPS-induced oxidative stress markers. Values represented as means ± SD; ∗ P
Figure Legend Snippet: Apigenin attenuates LPS-induced cardiac NF κ B nuclear translocation and oxidative damage. (a) Western blot analyses of NF κ B (p65) in nuclear and cytoplasmic fractions from heart lysates. Histone H3 was used as a nuclear control whereas tubulin was used as a cytoplasmic control. (b) Cardiac oxidative markers protein nitration and carbonyl content measured by quantitative ELISA. All markers were significantly increased in LPS-treated mice. Apigenin significantly reduced LPS-induced oxidative stress markers. Values represented as means ± SD; ∗ P

Techniques Used: Translocation Assay, Western Blot, Nitration, Enzyme-linked Immunosorbent Assay, Mouse Assay

25) Product Images from "Disruption of VEGF Mediated Flk‐1 Signaling Leads to a Gradual Loss of Vessel Health and Cardiac Function During Myocardial Infarction: Potential Therapy With Pellino‐1"

Article Title: Disruption of VEGF Mediated Flk‐1 Signaling Leads to a Gradual Loss of Vessel Health and Cardiac Function During Myocardial Infarction: Potential Therapy With Pellino‐1

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.117.007601

Transfection efficiency. A, Bar graph shows the expression of Ad‐LacZ and Ad‐Peli1 in control heart tissues by Western blot analysis 4 days after injection (n=3/group). B, Representative Western blot analysis of Peli1expression, 4 days after MI in CD 1 in mice. C, Bar graphs represent Peli1 expression normalized to GAPDH (n=3/group). Peli1 overexpression increased the phosphorylation of Akt, eNOS , MK 2, and IκBα after MI . D, Western blot analysis of p‐Akt and Akt (24 hours after MI ). E, Bar graphs represent p‐Akt levels normalized to total Akt levels (n=3/group). F, Western blot analysis of p‐ eNOS and eNOS (24 hours after MI ). G, Bar graphs represent p‐ eNOS levels normalized to total p‐ eNOS levels (n=4‐6/group). H, Western blot analysis of p‐ MK 2 and MK 2 (24 hours after MI ). I, The bar graphs represent p‐ MK 2 levels normalized to total MK 2 levels (n=3/group). J, Western blot analysis of p‐IκBα and IκBα (24 hours after MI ). K, Bar graphs represent p‐IκBα levels normalized to total IκBα levels (n=3/group). L through O, Western blot analyses of the β‐catenin expression in both cytosolic (L) and nuclear (N) fraction, 24 hours after MI . M, Bar graphs represent cytosolic β‐catenin levels normalized to GAPDH levels (n=5‐7/group). O, Bar graphs represent β‐catenin levels normalized to histone H3 levels. The Ad‐Peli1 MI group showed reduced levels of p‐Akt, p‐ eNOS , p‐ MK 2, p‐IκBα, and β‐catenin translocation compared with the Ad‐Lac ZMI group. Values are mean±SEM. * P
Figure Legend Snippet: Transfection efficiency. A, Bar graph shows the expression of Ad‐LacZ and Ad‐Peli1 in control heart tissues by Western blot analysis 4 days after injection (n=3/group). B, Representative Western blot analysis of Peli1expression, 4 days after MI in CD 1 in mice. C, Bar graphs represent Peli1 expression normalized to GAPDH (n=3/group). Peli1 overexpression increased the phosphorylation of Akt, eNOS , MK 2, and IκBα after MI . D, Western blot analysis of p‐Akt and Akt (24 hours after MI ). E, Bar graphs represent p‐Akt levels normalized to total Akt levels (n=3/group). F, Western blot analysis of p‐ eNOS and eNOS (24 hours after MI ). G, Bar graphs represent p‐ eNOS levels normalized to total p‐ eNOS levels (n=4‐6/group). H, Western blot analysis of p‐ MK 2 and MK 2 (24 hours after MI ). I, The bar graphs represent p‐ MK 2 levels normalized to total MK 2 levels (n=3/group). J, Western blot analysis of p‐IκBα and IκBα (24 hours after MI ). K, Bar graphs represent p‐IκBα levels normalized to total IκBα levels (n=3/group). L through O, Western blot analyses of the β‐catenin expression in both cytosolic (L) and nuclear (N) fraction, 24 hours after MI . M, Bar graphs represent cytosolic β‐catenin levels normalized to GAPDH levels (n=5‐7/group). O, Bar graphs represent β‐catenin levels normalized to histone H3 levels. The Ad‐Peli1 MI group showed reduced levels of p‐Akt, p‐ eNOS , p‐ MK 2, p‐IκBα, and β‐catenin translocation compared with the Ad‐Lac ZMI group. Values are mean±SEM. * P

Techniques Used: Transfection, Expressing, Western Blot, Injection, Mouse Assay, Over Expression, Translocation Assay

26) Product Images from "Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway"

Article Title: Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway

Journal: EMBO Reports

doi: 10.1093/embo-reports/kvf068

Fig. 1. Human Asf1 acts synergistically with CAF-1 to assemble nucleosomes during NER. ( A ) UV-damaged DNA substrate was incubated in a human cell-free system alone or complemented with core histones, CAF-1 (16 ng) or nuclear extract (2.5 µg protein). To these reactions, Asf1a or Asf1b (60 ng +, 250 ng + ) was added. Lane 1 contains untreated UV-damaged DNA. Radiolabel incorporation due to repair synthesis was visualized by autoradiography (labeled DNA) and total DNA by ethidium bromide staining (total DNA). Relaxed/nicked circular DNA (Ir/II) and supercoiled DNA (I) are indicated. ( B ) Analysis by MNase digestion of nucleosomal arrays formed on repaired DNA (labeled DNA) in a human cell-free system alone or complemented with histones, CAF-1 (16 ng +, 48 ng + ), Asf1a or Asf1b (60 ng) or nuclear extract as control (7.5 µg protein). Three digestion times (45 s and 3 and 6 min) are shown for each reaction. Positions corresponding to mono-, di-, tri- and tetra-nucleosomal DNA are indicated. Nuclear extract produced larger MNase digestion products, suggesting the involvement of additional factors in this extract. ( C ) Reactions as in (A) but to which Asf1a, Asf1b (60 ng) or nucleoplasmin (60 ng and 120 ng) was added.
Figure Legend Snippet: Fig. 1. Human Asf1 acts synergistically with CAF-1 to assemble nucleosomes during NER. ( A ) UV-damaged DNA substrate was incubated in a human cell-free system alone or complemented with core histones, CAF-1 (16 ng) or nuclear extract (2.5 µg protein). To these reactions, Asf1a or Asf1b (60 ng +, 250 ng + ) was added. Lane 1 contains untreated UV-damaged DNA. Radiolabel incorporation due to repair synthesis was visualized by autoradiography (labeled DNA) and total DNA by ethidium bromide staining (total DNA). Relaxed/nicked circular DNA (Ir/II) and supercoiled DNA (I) are indicated. ( B ) Analysis by MNase digestion of nucleosomal arrays formed on repaired DNA (labeled DNA) in a human cell-free system alone or complemented with histones, CAF-1 (16 ng +, 48 ng + ), Asf1a or Asf1b (60 ng) or nuclear extract as control (7.5 µg protein). Three digestion times (45 s and 3 and 6 min) are shown for each reaction. Positions corresponding to mono-, di-, tri- and tetra-nucleosomal DNA are indicated. Nuclear extract produced larger MNase digestion products, suggesting the involvement of additional factors in this extract. ( C ) Reactions as in (A) but to which Asf1a, Asf1b (60 ng) or nucleoplasmin (60 ng and 120 ng) was added.

Techniques Used: Incubation, Autoradiography, Labeling, Staining, Produced

27) Product Images from "Endoplasmic Reticulum Stress Induction of the Grp78/BiP Promoter: Activating Mechanisms Mediated by YY1 and Its Interactive Chromatin Modifiers"

Article Title: Endoplasmic Reticulum Stress Induction of the Grp78/BiP Promoter: Activating Mechanisms Mediated by YY1 and Its Interactive Chromatin Modifiers

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.11.4529-4540.2005

Model of ER stress-inducible changes in transcription factor occupancy and chromatin remodeling of the Grp78 promoter. In nonstressed cells, NF-Y is in contact with the Grp78 promoter. Upon ER stress, while NF-Y binding remains intact and TFII-I binding is enhanced, ATF6 is cleaved to produce a nuclear form, ATF6(N), which associates with YY1 and enhances its binding to the Grp78 promoter. The YY1-interacting proteins PRMT1 and p300 are also recruited to the Grp78 promoter. Additional chromatin changes of histone H4 include acetylation and arginine 3 methylation.
Figure Legend Snippet: Model of ER stress-inducible changes in transcription factor occupancy and chromatin remodeling of the Grp78 promoter. In nonstressed cells, NF-Y is in contact with the Grp78 promoter. Upon ER stress, while NF-Y binding remains intact and TFII-I binding is enhanced, ATF6 is cleaved to produce a nuclear form, ATF6(N), which associates with YY1 and enhances its binding to the Grp78 promoter. The YY1-interacting proteins PRMT1 and p300 are also recruited to the Grp78 promoter. Additional chromatin changes of histone H4 include acetylation and arginine 3 methylation.

Techniques Used: Binding Assay, Methylation

28) Product Images from "Inflamed macrophage microvesicles induce insulin resistance in human adipocytes"

Article Title: Inflamed macrophage microvesicles induce insulin resistance in human adipocytes

Journal: Nutrition & Metabolism

doi: 10.1186/s12986-015-0016-3

Effect of macrophage-derived MVs on insulin-stimulated activation of NF-κB in human adipocytes. a , b . Relative protein levels of nuclear NF-κB p65 in human mature adipocytes ( a ) and differentiated cells ( b ) were analyzed by western blotting. Human adipocytes were treated with indicated MVs for 24 h, and then incubated with 0, 1, 10, or 100 nM of insulin for 20 min. NF-κB p65 and histone H3 levels in nuclear lysates were determined by immunoblotting. Quantification of NF-κB p65 protein level is shown below. Western blot images were analyzed using Bandscan software, and statistical analysis is presented below. * P
Figure Legend Snippet: Effect of macrophage-derived MVs on insulin-stimulated activation of NF-κB in human adipocytes. a , b . Relative protein levels of nuclear NF-κB p65 in human mature adipocytes ( a ) and differentiated cells ( b ) were analyzed by western blotting. Human adipocytes were treated with indicated MVs for 24 h, and then incubated with 0, 1, 10, or 100 nM of insulin for 20 min. NF-κB p65 and histone H3 levels in nuclear lysates were determined by immunoblotting. Quantification of NF-κB p65 protein level is shown below. Western blot images were analyzed using Bandscan software, and statistical analysis is presented below. * P

Techniques Used: Derivative Assay, Activation Assay, Western Blot, Incubation, Software

Blocking of NF-κB reverse the inhibitory effect of M1 MVs on pAkt level and GLUT4 translocation in human adipocytes. a , d . Expression level of nuclear NF-κB p65 in human mature adipocytes ( a ) and differentiated cells ( d ) were analyzed by western blotting. Adipocytes were pretreated with or without NF-κB specific inhibitor (Bay, 10 μM) for 2 h, then incubated with M1 MVs for 24 h. Nuclear lysates were analyzed for NF-κB p65 and histone H3 level after insulin stimulation for a further 20 min. Quantification of NF-κB p65 protein level is presented below. b , e . Protein level of pAkt in human mature adipocytes ( b ) and differentiated adipocytes ( e ), after treatment as in a . Western blot images were analyzed using Bandscan software, and statistical analysis is presented below. c , f . The levels of GLUT4 in plasma membrane (PM) fractions of human mature adipocytes ( c ) and differentiated adipocytes ( f ) were determined by western blotting. Cells were treated as in A, and PM fractions were obtained and subjected to immunoblotting with the indicated antibodies. Quantification of the GLUT4 protein level is shown below. * P
Figure Legend Snippet: Blocking of NF-κB reverse the inhibitory effect of M1 MVs on pAkt level and GLUT4 translocation in human adipocytes. a , d . Expression level of nuclear NF-κB p65 in human mature adipocytes ( a ) and differentiated cells ( d ) were analyzed by western blotting. Adipocytes were pretreated with or without NF-κB specific inhibitor (Bay, 10 μM) for 2 h, then incubated with M1 MVs for 24 h. Nuclear lysates were analyzed for NF-κB p65 and histone H3 level after insulin stimulation for a further 20 min. Quantification of NF-κB p65 protein level is presented below. b , e . Protein level of pAkt in human mature adipocytes ( b ) and differentiated adipocytes ( e ), after treatment as in a . Western blot images were analyzed using Bandscan software, and statistical analysis is presented below. c , f . The levels of GLUT4 in plasma membrane (PM) fractions of human mature adipocytes ( c ) and differentiated adipocytes ( f ) were determined by western blotting. Cells were treated as in A, and PM fractions were obtained and subjected to immunoblotting with the indicated antibodies. Quantification of the GLUT4 protein level is shown below. * P

Techniques Used: Blocking Assay, Translocation Assay, Expressing, Western Blot, Incubation, Software

29) Product Images from "Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane"

Article Title: Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv181

Lamin A/C-associated pY19-Cav-2 in the INM regulates histone H3 modifications at the nuclear periphery in response to insulin. ( A and B ) Insulin-induced disassembly of H3K9me3 and assembly of H3K9ac and H3K18ac at the nuclear periphery composed of lamin A/C and Cav-2 in the INM. Hirc-B cells were treated with or without 100 nM insulin for 30 min, stained with anti-H3K9me3 ( A , a and B , a ), anti-AcH3 ( A , b ), anti-H3K9ac ( A , c and B , b ), anti-H3K18ac ( A , d and B , c ) or anti-H3K4ac ( A , e ) antibody followed by TRITC-conjugated antibody and with anti-lamin A/C ( A ) or anti-Cav-2 ( B ) antibody followed by FITC-conjugated antibody, and analyzed by confocal microscopy (mean ± SE, n = 3). The images on the right show magnifications of the areas framed in the Merge images. Scale bars, 20 μm. n.s., nonsignificant. ( C ) Regulation of insulin-induced disassembly of H3K9me3 and assembly of H3K9ac, H3K18ac and H3K27ac by lamin A/C-associated pY19-Cav-2. Whole cell lysates from control shRNA vector-expressed Hirc-B cells or GFP vector-expressed Cav-2 shRNA-, Cav-2-GFP-expressed Cav-2 shRNA-, Δ47–86-Cav-2-GFP-expressed Cav-2 shRNA- or Y19A-Cav-2-GFP-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to immunoblotting ( n = 4).
Figure Legend Snippet: Lamin A/C-associated pY19-Cav-2 in the INM regulates histone H3 modifications at the nuclear periphery in response to insulin. ( A and B ) Insulin-induced disassembly of H3K9me3 and assembly of H3K9ac and H3K18ac at the nuclear periphery composed of lamin A/C and Cav-2 in the INM. Hirc-B cells were treated with or without 100 nM insulin for 30 min, stained with anti-H3K9me3 ( A , a and B , a ), anti-AcH3 ( A , b ), anti-H3K9ac ( A , c and B , b ), anti-H3K18ac ( A , d and B , c ) or anti-H3K4ac ( A , e ) antibody followed by TRITC-conjugated antibody and with anti-lamin A/C ( A ) or anti-Cav-2 ( B ) antibody followed by FITC-conjugated antibody, and analyzed by confocal microscopy (mean ± SE, n = 3). The images on the right show magnifications of the areas framed in the Merge images. Scale bars, 20 μm. n.s., nonsignificant. ( C ) Regulation of insulin-induced disassembly of H3K9me3 and assembly of H3K9ac, H3K18ac and H3K27ac by lamin A/C-associated pY19-Cav-2. Whole cell lysates from control shRNA vector-expressed Hirc-B cells or GFP vector-expressed Cav-2 shRNA-, Cav-2-GFP-expressed Cav-2 shRNA-, Δ47–86-Cav-2-GFP-expressed Cav-2 shRNA- or Y19A-Cav-2-GFP-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to immunoblotting ( n = 4).

Techniques Used: Staining, Confocal Microscopy, shRNA, Plasmid Preparation

30) Product Images from "Tubular β-catenin and FoxO3 interactions protect in chronic kidney disease"

Article Title: Tubular β-catenin and FoxO3 interactions protect in chronic kidney disease

Journal: JCI Insight

doi: 10.1172/jci.insight.135454

Oxidative stress reduces β-catenin/LEF/TCF-dependent signaling and augments β-catenin/FoxO signaling. ( A ) PT cells were treated with varying doses of Wnt3a either in control conditions (PT medium as described in Methods) or with oxidative stress (H 2 O 2 100 μM in serum-free DMEM/F12) for 16 hours, and Axin2 transcripts were measured by qPCR and normalized to Gapdh . ( B ) PT cells stably transfected with a Topflash reporter construct (see Methods) were treated with various doses of Wnt3a in either control or oxidative stress medium (H 2 O 2 100 μM in serum-free DMEM/F12). A luminometer measured the TCF/LEF-dependent activity (Steady Glo), which was normalized to cell number by using Cell Titer assay. For both A and B , Holm-Šídák multiple-comparisons test was used. ( C ) Cells treated with AA for 5 days showed increased oxidative stress reflected by increased nitrotyrosine on immunoblots. PT cells were treated with AA (30 μM), Wnt3a (10 ng/mL) was added during the last 48 hours, and nuclei were isolated (see Methods) and immunoblotted for FoxO1, FoxO3, or histone H3 for loading ( D ), and the results from 3 separate experiments were quantified ( E ). ( F ) PT cells were treated ± Wnt3a (10 ng/mL) and oxidative stress (H 2 O 2 100 μM) for 16 hours, and then nuclei were isolated and coimmunoprecipitation was performed. Nuclear isolates had either β-catenin pull-down or IgG control, then were immunoblotted with FoxO3, and histone H3 was used for loading control of nuclear input. The nuclear input was also immunoblotted with α-tubulin, to assess for nuclear purity, and β-catenin. Levels of FoxO3 from the coimmunoprecipitation, normalized to histone H3 (nuclear input), were quantified from 3 separate experiments ( G ), as were nuclear β-catenin levels ( H ). Student’s t test was used for statistical analyses in G and H , and ANOVA was used for multiple comparisons in E with * P
Figure Legend Snippet: Oxidative stress reduces β-catenin/LEF/TCF-dependent signaling and augments β-catenin/FoxO signaling. ( A ) PT cells were treated with varying doses of Wnt3a either in control conditions (PT medium as described in Methods) or with oxidative stress (H 2 O 2 100 μM in serum-free DMEM/F12) for 16 hours, and Axin2 transcripts were measured by qPCR and normalized to Gapdh . ( B ) PT cells stably transfected with a Topflash reporter construct (see Methods) were treated with various doses of Wnt3a in either control or oxidative stress medium (H 2 O 2 100 μM in serum-free DMEM/F12). A luminometer measured the TCF/LEF-dependent activity (Steady Glo), which was normalized to cell number by using Cell Titer assay. For both A and B , Holm-Šídák multiple-comparisons test was used. ( C ) Cells treated with AA for 5 days showed increased oxidative stress reflected by increased nitrotyrosine on immunoblots. PT cells were treated with AA (30 μM), Wnt3a (10 ng/mL) was added during the last 48 hours, and nuclei were isolated (see Methods) and immunoblotted for FoxO1, FoxO3, or histone H3 for loading ( D ), and the results from 3 separate experiments were quantified ( E ). ( F ) PT cells were treated ± Wnt3a (10 ng/mL) and oxidative stress (H 2 O 2 100 μM) for 16 hours, and then nuclei were isolated and coimmunoprecipitation was performed. Nuclear isolates had either β-catenin pull-down or IgG control, then were immunoblotted with FoxO3, and histone H3 was used for loading control of nuclear input. The nuclear input was also immunoblotted with α-tubulin, to assess for nuclear purity, and β-catenin. Levels of FoxO3 from the coimmunoprecipitation, normalized to histone H3 (nuclear input), were quantified from 3 separate experiments ( G ), as were nuclear β-catenin levels ( H ). Student’s t test was used for statistical analyses in G and H , and ANOVA was used for multiple comparisons in E with * P

Techniques Used: Real-time Polymerase Chain Reaction, Stable Transfection, Transfection, Construct, Activity Assay, Titer Assay, Western Blot, Isolation

31) Product Images from "PARP1 inhibitors trigger innate immunity via PARP1 trapping-induced DNA damage response"

Article Title: PARP1 inhibitors trigger innate immunity via PARP1 trapping-induced DNA damage response

Journal: bioRxiv

doi: 10.1101/2020.07.12.199349

The PARP1 protein is required for PARPi-induced innate immune signaling. (A) PARPi-induced PARP1 trapping in wild-type (WT) and PARP1 knockout (KO) HeLa cells. Cell were also treated with or without Talazoparib (10 μM for 72 hrs). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ****p
Figure Legend Snippet: The PARP1 protein is required for PARPi-induced innate immune signaling. (A) PARPi-induced PARP1 trapping in wild-type (WT) and PARP1 knockout (KO) HeLa cells. Cell were also treated with or without Talazoparib (10 μM for 72 hrs). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ****p

Techniques Used: Knock-Out, Isolation

PARP1 trapping is the major contributor of PARPi-induced innate immune signaling. (A) The level of trapped PARP1 in HeLa cells treated with or without the indicated PARPi (10 μM for 72 hrs). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. *p
Figure Legend Snippet: PARP1 trapping is the major contributor of PARPi-induced innate immune signaling. (A) The level of trapped PARP1 in HeLa cells treated with or without the indicated PARPi (10 μM for 72 hrs). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. *p

Techniques Used: Isolation

PARP1 degraders abolish PARP1-trapping induced innate immune signaling. (A) The level of PARP1 in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 μM for 72 hrs). Whole cell lysates were probed using the indicated antibodies. GAPDH was used as the loading control. (B) The level of trapped PARP1 in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 μM for 72 hrs). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ***p
Figure Legend Snippet: PARP1 degraders abolish PARP1-trapping induced innate immune signaling. (A) The level of PARP1 in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 μM for 72 hrs). Whole cell lysates were probed using the indicated antibodies. GAPDH was used as the loading control. (B) The level of trapped PARP1 in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 μM for 72 hrs). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ***p

Techniques Used: Isolation

PARPi induces the innate immune response via the cGAS-STING pathway. (A) The level of trapped PARP1 in HeLa cells treated with or without Talazoparib (10 μM for 72 hrs). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with unpaired Student’s t-test. ***p
Figure Legend Snippet: PARPi induces the innate immune response via the cGAS-STING pathway. (A) The level of trapped PARP1 in HeLa cells treated with or without Talazoparib (10 μM for 72 hrs). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with unpaired Student’s t-test. ***p

Techniques Used: Isolation

32) Product Images from "Holocarboxylase Synthetase 1 Physically Interacts with Histone H3 in Arabidopsis"

Article Title: Holocarboxylase Synthetase 1 Physically Interacts with Histone H3 in Arabidopsis

Journal: Scientifica

doi: 10.1155/2013/983501

Avidin precipitation assays. The protein samples were analyzed by 15% SDS-PAGE. I 125 -streptavidin (left panel) and histone H3 antibody (right panel) were used in the western blots. Input: total protein extracted from Arabidopsis . B: blank control, where avidin beads were used only with release buffer. Sup: the supernatant after the avidin beads were incubated with Arabidopsis lysate overnight. W1/2/3: the supernatants for the first, second, and third washes separately after the incubation. AP: precipitation of total protein preformed with avidin beads. ACC1: homomeric acetyl-CoA carboxylase. MCC: methylcrotonyl-CoA carboxylase. BCCP1: biotin carboxyl carrier protein1, which is part of the heteromeric acetyl-CoA carboxylase. BCCP2: biotin carboxyl carrier protein2, which is part of the heteromeric acetyl-CoA carboxylase. Experiments were conducted in triplicate.
Figure Legend Snippet: Avidin precipitation assays. The protein samples were analyzed by 15% SDS-PAGE. I 125 -streptavidin (left panel) and histone H3 antibody (right panel) were used in the western blots. Input: total protein extracted from Arabidopsis . B: blank control, where avidin beads were used only with release buffer. Sup: the supernatant after the avidin beads were incubated with Arabidopsis lysate overnight. W1/2/3: the supernatants for the first, second, and third washes separately after the incubation. AP: precipitation of total protein preformed with avidin beads. ACC1: homomeric acetyl-CoA carboxylase. MCC: methylcrotonyl-CoA carboxylase. BCCP1: biotin carboxyl carrier protein1, which is part of the heteromeric acetyl-CoA carboxylase. BCCP2: biotin carboxyl carrier protein2, which is part of the heteromeric acetyl-CoA carboxylase. Experiments were conducted in triplicate.

Techniques Used: Avidin-Biotin Assay, SDS Page, Western Blot, Incubation

2D gel analysis. (a) Histone proteins were enriched from Arabidopsis young seedling lysate and stained with Coomassie Blue. The same amount of total and histone-enriched proteins was separated by 15% SDS-PAGE and the western blot results showed that histone proteins were enriched from Arabidopsis young seedling lysate. W: the total isolated proteins. H: the histone-enriched proteins. Ct: commercial calf thymus histones (Sigma). (b) The histone-enriched proteins were analyzed by 20% SDS-PAGE with pH gradient from 9 to 12. The biotin signals that concentrate at pH 9 versus the core-histones signals that distribute between pH 10 to 11. Experiments were conducted in triplicate.
Figure Legend Snippet: 2D gel analysis. (a) Histone proteins were enriched from Arabidopsis young seedling lysate and stained with Coomassie Blue. The same amount of total and histone-enriched proteins was separated by 15% SDS-PAGE and the western blot results showed that histone proteins were enriched from Arabidopsis young seedling lysate. W: the total isolated proteins. H: the histone-enriched proteins. Ct: commercial calf thymus histones (Sigma). (b) The histone-enriched proteins were analyzed by 20% SDS-PAGE with pH gradient from 9 to 12. The biotin signals that concentrate at pH 9 versus the core-histones signals that distribute between pH 10 to 11. Experiments were conducted in triplicate.

Techniques Used: Two-Dimensional Gel Electrophoresis, Staining, SDS Page, Western Blot, Isolation

HCS1 interacts with Arabidopsis histone H3 directly in vitro . E. coli lines that contain GST-HCS1, His-H3, or GST construct were induced to express these genes. After induction, total protein was extracted from the cell lines. Recombinant His-histone H3 protein was purified from E. coli by using metal affinity chromatography. Recombinant GST-HCS1 or GST alone was purified from E. coli by using glutathione-sepharose beads. Proteins were subjected to GST pull-down assays with GST-HCS1 or GST alone and probed by western blot to detect whether there was an interaction between HCS1 and histone H3. (a) Proteins were analyzed by 15% SDS-PAGE and stained with Coomassie Blue. I: total proteins extracted from E. coli containing a GST-HCS1 or a His-H3 construct. C: total proteins extracted from control E. coli (not containing any vector). P: recombinant GST-HCS1 or recombinant His-H3 proteins. (b) Western blot of proteins after GST pull-down to detect whether there was an interaction between HCS1 and histone H3. GST-HSC1 fusion protein and GST were detected with GST antibody; Histone H3 was detected with corresponding antibody. GST-HCS1: the proteins released from the beads coupled with GST-HCS1 after being incubated with His-histone H3 proteins; Histone H3 pulled down by GST-HCS1 was detected. GST: the proteins released from the beads coupled with GST control after being incubated with His-histone H3 proteins; no histone H3 was detected. Input His-H3: 10% of the total input recombinant His-histone H3 proteins; Histone H3 was strongly detected as expected. Experiments were conducted in triplicate.
Figure Legend Snippet: HCS1 interacts with Arabidopsis histone H3 directly in vitro . E. coli lines that contain GST-HCS1, His-H3, or GST construct were induced to express these genes. After induction, total protein was extracted from the cell lines. Recombinant His-histone H3 protein was purified from E. coli by using metal affinity chromatography. Recombinant GST-HCS1 or GST alone was purified from E. coli by using glutathione-sepharose beads. Proteins were subjected to GST pull-down assays with GST-HCS1 or GST alone and probed by western blot to detect whether there was an interaction between HCS1 and histone H3. (a) Proteins were analyzed by 15% SDS-PAGE and stained with Coomassie Blue. I: total proteins extracted from E. coli containing a GST-HCS1 or a His-H3 construct. C: total proteins extracted from control E. coli (not containing any vector). P: recombinant GST-HCS1 or recombinant His-H3 proteins. (b) Western blot of proteins after GST pull-down to detect whether there was an interaction between HCS1 and histone H3. GST-HSC1 fusion protein and GST were detected with GST antibody; Histone H3 was detected with corresponding antibody. GST-HCS1: the proteins released from the beads coupled with GST-HCS1 after being incubated with His-histone H3 proteins; Histone H3 pulled down by GST-HCS1 was detected. GST: the proteins released from the beads coupled with GST control after being incubated with His-histone H3 proteins; no histone H3 was detected. Input His-H3: 10% of the total input recombinant His-histone H3 proteins; Histone H3 was strongly detected as expected. Experiments were conducted in triplicate.

Techniques Used: In Vitro, Construct, Recombinant, Purification, Affinity Chromatography, Western Blot, SDS Page, Staining, Plasmid Preparation, Incubation

33) Product Images from "SMYD3 promotes the epithelial–mesenchymal transition in breast cancer"

Article Title: SMYD3 promotes the epithelial–mesenchymal transition in breast cancer

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky1221

SMYD3 assists SMAD3 recruitment to the chromatin. ( A ) SMAD2/3 and SMYD3 association to regulatory regions of EMT markers was analyzed by ChIP qPCR, in ShScramble and ShSMYD3 MCF10A cells treated with 5ng/ml TGFβ for 24 h. Data are expressed as fold enrichment relative to ShScramble cells and represent means±SD. Statistical analysis was performed with Student's t test. n = 4, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ( B ) SMAD2/3 and SMYD3 association to EMT markers was analyzed by ChIP qPCR, in MCF10A cells co-treated with 5ng/ml TGFβ and 10 μM BCI121 for 24 h. Data are expressed as fold enrichment relative to control cells. Data represent means±SD. Statistical analysis was performed with unpaired Student's t test. n = 4, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ( C ) immunofluorescence microscopy of shSMYD3 or shScramble NMuMG cells treated with 10 ng/ml TGFβ for 2 h. The staining for the phosphorylated SMAD3 (P-SMAD3) (left panel), for DAPI (center panel) and the merge of the two stainings (right panel) are shown. Scale bar = 10 μm. ( D ) Western-blot analysis of P-SMAD3 and SMAD2/3 levels in shSMYD3 or shScramble NMuMG cells, treated with 10 ng/ml TGFβ for for 30 min. Relative band intensity, normalized over total SMAD2/3 proteins, is indicated. ( E ) Western-blot analysis of cytoplasmic (C) and nuclear (N) P-SMAD3 in shSMYD3 or shScramble NMuMG cells treated with 10 ng/ml TGFβ for 30 min. GAPDH was used as cytoplasmic marker, histone H3 as nuclear marker. ( F ) ChIP assay was performed to evaluate SMYD3, SMAD2/3, RNAPolII, H3K9me3, H3K9Ac, H3K27me3 occupancy at the Snail1 promoter, in NMuMG cells treated with 10ng/ml TGFβ for 6 h. Data are normalized on IgG and represent means ± SD. Statistical analysis was performed with Student's t test. n = 3, * P ≤ 0.05, ** P ≤ 0.01. ( G ) ChIP assay was performed to evaluate SMYD3 and SMAD2/3 occupancy at the Snail1 promoter, in NMuMG cells obtained as in Figure 3F , treated with 10ng/ml TGFβ for 6 h. SiS refers to SiSMYD3 cells. Data are normalized on IgG and represent means±SD. Statistical analysis was performed with unpaired Student's t test. n = 3, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
Figure Legend Snippet: SMYD3 assists SMAD3 recruitment to the chromatin. ( A ) SMAD2/3 and SMYD3 association to regulatory regions of EMT markers was analyzed by ChIP qPCR, in ShScramble and ShSMYD3 MCF10A cells treated with 5ng/ml TGFβ for 24 h. Data are expressed as fold enrichment relative to ShScramble cells and represent means±SD. Statistical analysis was performed with Student's t test. n = 4, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ( B ) SMAD2/3 and SMYD3 association to EMT markers was analyzed by ChIP qPCR, in MCF10A cells co-treated with 5ng/ml TGFβ and 10 μM BCI121 for 24 h. Data are expressed as fold enrichment relative to control cells. Data represent means±SD. Statistical analysis was performed with unpaired Student's t test. n = 4, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ( C ) immunofluorescence microscopy of shSMYD3 or shScramble NMuMG cells treated with 10 ng/ml TGFβ for 2 h. The staining for the phosphorylated SMAD3 (P-SMAD3) (left panel), for DAPI (center panel) and the merge of the two stainings (right panel) are shown. Scale bar = 10 μm. ( D ) Western-blot analysis of P-SMAD3 and SMAD2/3 levels in shSMYD3 or shScramble NMuMG cells, treated with 10 ng/ml TGFβ for for 30 min. Relative band intensity, normalized over total SMAD2/3 proteins, is indicated. ( E ) Western-blot analysis of cytoplasmic (C) and nuclear (N) P-SMAD3 in shSMYD3 or shScramble NMuMG cells treated with 10 ng/ml TGFβ for 30 min. GAPDH was used as cytoplasmic marker, histone H3 as nuclear marker. ( F ) ChIP assay was performed to evaluate SMYD3, SMAD2/3, RNAPolII, H3K9me3, H3K9Ac, H3K27me3 occupancy at the Snail1 promoter, in NMuMG cells treated with 10ng/ml TGFβ for 6 h. Data are normalized on IgG and represent means ± SD. Statistical analysis was performed with Student's t test. n = 3, * P ≤ 0.05, ** P ≤ 0.01. ( G ) ChIP assay was performed to evaluate SMYD3 and SMAD2/3 occupancy at the Snail1 promoter, in NMuMG cells obtained as in Figure 3F , treated with 10ng/ml TGFβ for 6 h. SiS refers to SiSMYD3 cells. Data are normalized on IgG and represent means±SD. Statistical analysis was performed with unpaired Student's t test. n = 3, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunofluorescence, Microscopy, Staining, Western Blot, Marker

34) Product Images from "Chronic Ethanol Consumption Induces Global Hepatic Protein Hyperacetylation"

Article Title: Chronic Ethanol Consumption Induces Global Hepatic Protein Hyperacetylation

Journal: Alcoholism, clinical and experimental research

doi: 10.1111/j.1530-0277.2009.01091.x

Chronic ethanol treatment induces acetylation of nuclear, cytosolic, and membrane proteins. Liver homogenates from control (C) and ethanol (E) pair-fed rats were separated by differential centrifugation to prepare nuclei, cytosol, or total membranes (TM). ( A ) Fractions were immunoblotted with anti-acetylated lysine antibodies to detect hyperacetylated proteins (marked by arrows). Molecular weight standards are indicated on the left. ( B ) Fractions were immunoblotted for acetylated histone H3 (Ac-H3; a nuclear marker protein), tubulin (a cytosolic marker protein), and the basolateral resident protein, CE9 (a membrane marker protein) as indicated. The ethanol-induced increase in histone H3 acetylation is indicated in parentheses. The value is the average ± SEM from 3 independent sets of pair-fed animals.
Figure Legend Snippet: Chronic ethanol treatment induces acetylation of nuclear, cytosolic, and membrane proteins. Liver homogenates from control (C) and ethanol (E) pair-fed rats were separated by differential centrifugation to prepare nuclei, cytosol, or total membranes (TM). ( A ) Fractions were immunoblotted with anti-acetylated lysine antibodies to detect hyperacetylated proteins (marked by arrows). Molecular weight standards are indicated on the left. ( B ) Fractions were immunoblotted for acetylated histone H3 (Ac-H3; a nuclear marker protein), tubulin (a cytosolic marker protein), and the basolateral resident protein, CE9 (a membrane marker protein) as indicated. The ethanol-induced increase in histone H3 acetylation is indicated in parentheses. The value is the average ± SEM from 3 independent sets of pair-fed animals.

Techniques Used: Centrifugation, Molecular Weight, Marker

35) Product Images from "A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication"

Article Title: A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication

Journal: Science signaling

doi: 10.1126/scisignal.2003417

HIF-1α inhibits Cdc7-mediated phosphorylation and activation of the MCM helicase. ( A ) The chromatin fraction and whole-cell lysates of HCT116 cells exposed to 20 or 1% O 2 were isolated and analyzed with anti-Cdc7, HIF-1α, or histone H3 antibodies. ( B and C ) NIH-3T3 cells were untreated or treated with DFX (B) or DMOG (C) for 24 hours, after which the chromatin fraction was isolated. ( D ) HCT116 cells were left untreated, treated with DMOG, or exposed to 1% O 2 for 24 hours, and cell lysates were probed with the indicated antibodies. ( E ) The chromatin fraction from HCT116 cells exposed to 20 or 1% O 2 was probed with indicated antibodies. ( F ) HeLa cells were processed as in (D). ( G ) Lysates from HeLa, HCT116, and WT8 cells exposed to either 20 or 1% O 2 were probed with the indicated antibodies. ( H ) The chromatin fraction from HCT116 cells exposed to 20 or 1% O 2 was probed with indicated antibodies. ( I and J ) HCT116 cells were transfected with either empty shRNA vector or vector encoding shRNA against HIF-1α and exposed to DMOG (I) or 1% O 2 (J). The chromatin fraction was analyzed with the indicated antibodies. Data in (I) and (J) represent two independent experiments. Western blot data from three independent replicates were quantified for (A) to (H). Results are shown as means ± SEM. * P
Figure Legend Snippet: HIF-1α inhibits Cdc7-mediated phosphorylation and activation of the MCM helicase. ( A ) The chromatin fraction and whole-cell lysates of HCT116 cells exposed to 20 or 1% O 2 were isolated and analyzed with anti-Cdc7, HIF-1α, or histone H3 antibodies. ( B and C ) NIH-3T3 cells were untreated or treated with DFX (B) or DMOG (C) for 24 hours, after which the chromatin fraction was isolated. ( D ) HCT116 cells were left untreated, treated with DMOG, or exposed to 1% O 2 for 24 hours, and cell lysates were probed with the indicated antibodies. ( E ) The chromatin fraction from HCT116 cells exposed to 20 or 1% O 2 was probed with indicated antibodies. ( F ) HeLa cells were processed as in (D). ( G ) Lysates from HeLa, HCT116, and WT8 cells exposed to either 20 or 1% O 2 were probed with the indicated antibodies. ( H ) The chromatin fraction from HCT116 cells exposed to 20 or 1% O 2 was probed with indicated antibodies. ( I and J ) HCT116 cells were transfected with either empty shRNA vector or vector encoding shRNA against HIF-1α and exposed to DMOG (I) or 1% O 2 (J). The chromatin fraction was analyzed with the indicated antibodies. Data in (I) and (J) represent two independent experiments. Western blot data from three independent replicates were quantified for (A) to (H). Results are shown as means ± SEM. * P

Techniques Used: Activation Assay, Isolation, Transfection, shRNA, Plasmid Preparation, Western Blot

36) Product Images from "Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3"

Article Title: Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3

Journal: PLoS ONE

doi: 10.1371/journal.pone.0071518

The HER3 receptor is localized to the nucleus in its full-length form. A. HER3 is expressed in numerous cancer cell lines. Whole cell protein lysates were isolated from various breast, lung, HNSCC, and colon cancer cell lines. Lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin was used as a loading control. B. HER3 is localized to the nucleus in cancer cell lines. H226 R , SKBr3, MCF-7, and BT549 cells were harvested for whole cell (WC), non-nuclear (NN) and nuclear (Nuc) protein, fractionated on SDS-PAGE followed by immunoblotting for HER3. Clathrin, dynamin, calnexin, α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. C. Specificity of nuclear HER3 by siRNA. H226 R and SKBr3 cells were harvested for NN and Nuc protein 48 hr post treatment with siHER3 or non-targeting (NT) siRNA. Experimental procedure as in 1B. α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. D. Full-length HER3 is localized to the nucleus. H226 R and SKBr3 cells were harvested for WC, NN, and Nuc lysate. WC lysates were harvested 48 hr post treatment with siHER3. 250 ug of cell lysate was immunoprecipitated with an N-TERM HER3 antibody or human IgG control. The immunoprecipitates were fractionated on SDS-PAGE followed by immunoblotting for HER3 with a C-TERM antibody.
Figure Legend Snippet: The HER3 receptor is localized to the nucleus in its full-length form. A. HER3 is expressed in numerous cancer cell lines. Whole cell protein lysates were isolated from various breast, lung, HNSCC, and colon cancer cell lines. Lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin was used as a loading control. B. HER3 is localized to the nucleus in cancer cell lines. H226 R , SKBr3, MCF-7, and BT549 cells were harvested for whole cell (WC), non-nuclear (NN) and nuclear (Nuc) protein, fractionated on SDS-PAGE followed by immunoblotting for HER3. Clathrin, dynamin, calnexin, α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. C. Specificity of nuclear HER3 by siRNA. H226 R and SKBr3 cells were harvested for NN and Nuc protein 48 hr post treatment with siHER3 or non-targeting (NT) siRNA. Experimental procedure as in 1B. α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. D. Full-length HER3 is localized to the nucleus. H226 R and SKBr3 cells were harvested for WC, NN, and Nuc lysate. WC lysates were harvested 48 hr post treatment with siHER3. 250 ug of cell lysate was immunoprecipitated with an N-TERM HER3 antibody or human IgG control. The immunoprecipitates were fractionated on SDS-PAGE followed by immunoblotting for HER3 with a C-TERM antibody.

Techniques Used: Isolation, SDS Page, Immunoprecipitation

Nuclear HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain. A. HER3 knockdown decreases the activation of the cyclin D1 promoter. H226 R , SKBr3, and MCF-7 cells were incubated with non-targeting (NT) or HER3 siRNA for 24 hr followed by transfection with the 122 bp cyclin D1 promoter-luciferase and Tk- Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 6). Percent decreases in cyclin D1 promoter-luciferase activity were normalized to NT transfected cells. The graph is representative of three independent experiments. Luciferase lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. B. HER3 overexpression activates the cyclin D1 promoter. SCC6, HCC1954, SKBr3, and BT474 cells were transfected with HER3WT or control vector, 122 bp cyclin-D1-luciferase and Tk- Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1-promoter luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. Nuclear lysate was harvested from each cell line and fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for the Nuc fraction. C. HER3ΔB 1 ΔB 2 overexpression can prevent the activation of the cyclin D1 promoter while HER3DM remains functional. SCC6, BT474 and HCC1954 cells were transfected with HER3WT, HER3DM, HER3ΔB 1 ΔB 2 or control vector, the 122 bp cyclin-D1-luciferase and Tk- Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of seven independent experiments. Inset 1: CHOK1 cells were transfected with HER3WT, HER3ΔB 1 ΔB 2 or control vector for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. Inset 2: CHOK1 cells were transfected with HER3WT, HER3DM, HER3ΔB 1 ΔB 2 or control vector and stimulated for 40 min with 5 nM neuregulin-1. Whole cell lysate was fractionated on SDS-PAGE followed by immunoblotting for indicated proteins. D. HER3 knockdown decreases cyclin D1 expression. SCC6 and BT474 cells were incubated with NT or HER3 siRNA for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to NT transfected cells. The graph is representative of two independent experiments. E. The overexpression of HER3WT enhances cyclin D1 expression while HER3ΔB 1 ΔB 2 is hindered. SCC6 and BT474 cells were transfected with HER3WT, HER3ΔB 1 ΔB 2 or control vector for 72 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of four independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of Renilla luciferase. All data points are represented as mean +/− s.e.m. P
Figure Legend Snippet: Nuclear HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain. A. HER3 knockdown decreases the activation of the cyclin D1 promoter. H226 R , SKBr3, and MCF-7 cells were incubated with non-targeting (NT) or HER3 siRNA for 24 hr followed by transfection with the 122 bp cyclin D1 promoter-luciferase and Tk- Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 6). Percent decreases in cyclin D1 promoter-luciferase activity were normalized to NT transfected cells. The graph is representative of three independent experiments. Luciferase lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. B. HER3 overexpression activates the cyclin D1 promoter. SCC6, HCC1954, SKBr3, and BT474 cells were transfected with HER3WT or control vector, 122 bp cyclin-D1-luciferase and Tk- Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1-promoter luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. Nuclear lysate was harvested from each cell line and fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for the Nuc fraction. C. HER3ΔB 1 ΔB 2 overexpression can prevent the activation of the cyclin D1 promoter while HER3DM remains functional. SCC6, BT474 and HCC1954 cells were transfected with HER3WT, HER3DM, HER3ΔB 1 ΔB 2 or control vector, the 122 bp cyclin-D1-luciferase and Tk- Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of seven independent experiments. Inset 1: CHOK1 cells were transfected with HER3WT, HER3ΔB 1 ΔB 2 or control vector for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. Inset 2: CHOK1 cells were transfected with HER3WT, HER3DM, HER3ΔB 1 ΔB 2 or control vector and stimulated for 40 min with 5 nM neuregulin-1. Whole cell lysate was fractionated on SDS-PAGE followed by immunoblotting for indicated proteins. D. HER3 knockdown decreases cyclin D1 expression. SCC6 and BT474 cells were incubated with NT or HER3 siRNA for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to NT transfected cells. The graph is representative of two independent experiments. E. The overexpression of HER3WT enhances cyclin D1 expression while HER3ΔB 1 ΔB 2 is hindered. SCC6 and BT474 cells were transfected with HER3WT, HER3ΔB 1 ΔB 2 or control vector for 72 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of four independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of Renilla luciferase. All data points are represented as mean +/− s.e.m. P

Techniques Used: Activation Assay, Incubation, Transfection, Luciferase, Activity Assay, SDS Page, Over Expression, Plasmid Preparation, Functional Assay, Fractionation, Expressing, Real-time Polymerase Chain Reaction

An intracellular domain (ICD) mutant of HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain. A. HER3WT ICD (WT-ICD) can activate the cyclin D1 promoter while HER3ΔB 1 ΔB 2 -ICD (ΔB 1 ΔB 2 -ICD) is hindered. SCC6, BT474, and HCC1954 cells were transfected with WT-ICD, ΔB 1 ΔB 2 -ICD or control vector, the 122 bp cyclin D1-luciferase and Tk- Renilla reporter pl asmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. Inset 1: Illustration of both the WT-ICD and ΔB 1 ΔB 2 -ICD constructs. Inset 2: CHOK1 cells were transfected with WT-ICD and ΔB 1 ΔB 2 -ICD for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. B. The overexpression of WT-ICD can enhance cyclin D1 expression while ΔB 1 ΔB 2 -ICD is hindered. SCC6 and BT474 cells were transfected with WT-ICD, ΔB 1 ΔB 2 -ICD or control vector for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of three independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of Renilla luciferase. All data points are represented as mean +/− s.e.m. P
Figure Legend Snippet: An intracellular domain (ICD) mutant of HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain. A. HER3WT ICD (WT-ICD) can activate the cyclin D1 promoter while HER3ΔB 1 ΔB 2 -ICD (ΔB 1 ΔB 2 -ICD) is hindered. SCC6, BT474, and HCC1954 cells were transfected with WT-ICD, ΔB 1 ΔB 2 -ICD or control vector, the 122 bp cyclin D1-luciferase and Tk- Renilla reporter pl asmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. Inset 1: Illustration of both the WT-ICD and ΔB 1 ΔB 2 -ICD constructs. Inset 2: CHOK1 cells were transfected with WT-ICD and ΔB 1 ΔB 2 -ICD for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. B. The overexpression of WT-ICD can enhance cyclin D1 expression while ΔB 1 ΔB 2 -ICD is hindered. SCC6 and BT474 cells were transfected with WT-ICD, ΔB 1 ΔB 2 -ICD or control vector for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of three independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of Renilla luciferase. All data points are represented as mean +/− s.e.m. P

Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Luciferase, Construct, Fractionation, SDS Page, Over Expression, Expressing, Real-time Polymerase Chain Reaction

37) Product Images from "Quantitative Proteomic Analysis of Chromatin Reveals that Ctf18 Acts in the DNA Replication Checkpoint *"

Article Title: Quantitative Proteomic Analysis of Chromatin Reveals that Ctf18 Acts in the DNA Replication Checkpoint *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M110.005561

Increased chromatin binding of DNA replication proteins, the checkpoint kinase Mec1, and cohesin proteins in HU-treated ctf18 Δ cells. A , Plot shows log 2 ratios of all chromosome proteins identified and their summed peptide intensities. In this and subsequent plots, the marker symbols indicate significance scores for the changes observed, with green diamonds indicating the highly significant abnormalities and blue crosses changes less likely to be significant. ctf18 Δ cells were labeled with [ 13 C 6 ]-Lys. Strains used are SHY201 and TKY1. B , Schematic representation of replisome proteins, colored according to their altered chromatin loading. C , Western blot analysis confirms changed chromatin binding levels. Western blots show whole cell extract (WCE; lanes 1 and 2) and chromatin-enriched (Ch; lanes 3 and 4) fractions from strains with epitope-tagged proteins Rfa1–3HA, Ctf4–13Myc, PCNA-3Myc, or Psf2–13Myc. Loading of Ch fractions was adjusted to be appropriate for each protein analyzed. A dilution series of wild-type chromatin (lanes 5–8) allows the assembly of a standard plot for quantification. Strains used are TKY27, TKY33, TKY25, TKY31, Y1109, SHY164, TKY22, and TKY23. Top panel (Mcm2) shows TKY27 and TKY33. D , Histogram shows ctf18 Δ/WT ratios in Ch fraction for each protein, as measured by Western blots. Ratios were calculated based on signal intensities normalized against levels of histone H3 (see also Supplemental Fig. S3 ). E , Histogram shows ctf18 Δ/WT ratios in Ch fraction on log scale, as measured in SILAC analysis (open bars) and by Western blotting (filled bars). For Western blot analysis ratios, mean value and standard deviation (error bar) of Mcm2 from four experiments is shown. For the SILAC analysis ratios, mean values and standard deviations (error bars) are derived from two independent experiments.
Figure Legend Snippet: Increased chromatin binding of DNA replication proteins, the checkpoint kinase Mec1, and cohesin proteins in HU-treated ctf18 Δ cells. A , Plot shows log 2 ratios of all chromosome proteins identified and their summed peptide intensities. In this and subsequent plots, the marker symbols indicate significance scores for the changes observed, with green diamonds indicating the highly significant abnormalities and blue crosses changes less likely to be significant. ctf18 Δ cells were labeled with [ 13 C 6 ]-Lys. Strains used are SHY201 and TKY1. B , Schematic representation of replisome proteins, colored according to their altered chromatin loading. C , Western blot analysis confirms changed chromatin binding levels. Western blots show whole cell extract (WCE; lanes 1 and 2) and chromatin-enriched (Ch; lanes 3 and 4) fractions from strains with epitope-tagged proteins Rfa1–3HA, Ctf4–13Myc, PCNA-3Myc, or Psf2–13Myc. Loading of Ch fractions was adjusted to be appropriate for each protein analyzed. A dilution series of wild-type chromatin (lanes 5–8) allows the assembly of a standard plot for quantification. Strains used are TKY27, TKY33, TKY25, TKY31, Y1109, SHY164, TKY22, and TKY23. Top panel (Mcm2) shows TKY27 and TKY33. D , Histogram shows ctf18 Δ/WT ratios in Ch fraction for each protein, as measured by Western blots. Ratios were calculated based on signal intensities normalized against levels of histone H3 (see also Supplemental Fig. S3 ). E , Histogram shows ctf18 Δ/WT ratios in Ch fraction on log scale, as measured in SILAC analysis (open bars) and by Western blotting (filled bars). For Western blot analysis ratios, mean value and standard deviation (error bar) of Mcm2 from four experiments is shown. For the SILAC analysis ratios, mean values and standard deviations (error bars) are derived from two independent experiments.

Techniques Used: Binding Assay, Marker, Labeling, Western Blot, Standard Deviation, Derivative Assay

38) Product Images from "Yin Yang 1 Is a Repressor of Glutamate Transporter EAAT2, and It Mediates Manganese-Induced Decrease of EAAT2 Expression in Astrocytes"

Article Title: Yin Yang 1 Is a Repressor of Glutamate Transporter EAAT2, and It Mediates Manganese-Induced Decrease of EAAT2 Expression in Astrocytes

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01176-13

Mn increases YY1 expression. (A) Confocal image showing YY1 and GFAP expression in rat primary astrocytes. (B) After overnight transfection with the EAAT2 promoter vector, astrocytes were treated with Mn (0, 125, and 250 μM) for 6 h, followed by a luciferase assay. (C and D) Astrocytes were treated with Mn (with 250 μM for up to 3 h or for 3 h with up to 250 μM), followed by measurement of YY1 mRNA levels by qPCR (C) and conventional reverse transcription-PCR (D) using GAPDH as a control. (E) After treatment with Mn (250 μM), astrocytes were lysed, and YY1 protein levels were measured in whole-astrocyte lysates (top panel) or nuclear extract (bottom panel) by Western blotting. Equal amounts (30 μg) of cell lysates or nuclear extracts were loaded using β-actin and histone H3 as internal controls. *, P
Figure Legend Snippet: Mn increases YY1 expression. (A) Confocal image showing YY1 and GFAP expression in rat primary astrocytes. (B) After overnight transfection with the EAAT2 promoter vector, astrocytes were treated with Mn (0, 125, and 250 μM) for 6 h, followed by a luciferase assay. (C and D) Astrocytes were treated with Mn (with 250 μM for up to 3 h or for 3 h with up to 250 μM), followed by measurement of YY1 mRNA levels by qPCR (C) and conventional reverse transcription-PCR (D) using GAPDH as a control. (E) After treatment with Mn (250 μM), astrocytes were lysed, and YY1 protein levels were measured in whole-astrocyte lysates (top panel) or nuclear extract (bottom panel) by Western blotting. Equal amounts (30 μg) of cell lysates or nuclear extracts were loaded using β-actin and histone H3 as internal controls. *, P

Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot

HDAC inhibits the stimulatory p65 effects on EAAT2 promoter activity. (A and B) Astrocytes were cotransfected overnight with either or both p65 and HDAC1 or HDAC4 expression vectors or their empty vectors (control) along with the EAAT2 promoter vector, followed by a luciferase assay. (C) The co-IP of HDAC1 and p65 was carried out using the nuclear extracts prepared from control and Mn-treated cells. (D) The co-IP of HDAC1 and p65 was performed using nuclear extracts which were prepared from astrocytes transfected with scrambled control (sc siRNA) and YY1 siRNAs for 48 h. Nuclear extracts were also blotted for YY1 to confirm the efficiency of knockdown with YY1 siRNA. Histone H3 was used as a loading control. #, P
Figure Legend Snippet: HDAC inhibits the stimulatory p65 effects on EAAT2 promoter activity. (A and B) Astrocytes were cotransfected overnight with either or both p65 and HDAC1 or HDAC4 expression vectors or their empty vectors (control) along with the EAAT2 promoter vector, followed by a luciferase assay. (C) The co-IP of HDAC1 and p65 was carried out using the nuclear extracts prepared from control and Mn-treated cells. (D) The co-IP of HDAC1 and p65 was performed using nuclear extracts which were prepared from astrocytes transfected with scrambled control (sc siRNA) and YY1 siRNAs for 48 h. Nuclear extracts were also blotted for YY1 to confirm the efficiency of knockdown with YY1 siRNA. Histone H3 was used as a loading control. #, P

Techniques Used: Activity Assay, Expressing, Plasmid Preparation, Luciferase, Co-Immunoprecipitation Assay, Transfection

39) Product Images from "Mechanism of Growth Inhibition of Prostate Cancer Xenografts by Valproic Acid"

Article Title: Mechanism of Growth Inhibition of Prostate Cancer Xenografts by Valproic Acid

Journal: Journal of Biomedicine and Biotechnology

doi: 10.1155/2012/180363

Animals with prostate cancer xenografts were randomized into control and treatment arms. Animals in treatment arm received 0.4% w/v Valproic Acid (VPA) in drinking water. Animals were treated for 35 days before excision of tumors. Tissue Microarrays (TMAs) of xenografts were created and expression of various markers was studied. Representative images of xenograft sections from control and VPA-treated groups; LNCaP, C4-2, and DU-145 (scanned at 20x magnification using the APERIO imaging system) are shown. (a) Immunohistochemistry (IHC) staining for acetylated histone 3. (b) Staining of cell cycle regulators (p21, p27, cyclin D1). (c) Staining for differentiation markers (Cytokeratin 18 (CK18), androgen receptor (AR)). (d) Staining for markers of proliferation (Ki-67), apoptosis (TUNEL), and angiogenesis (mean vascular density, MVD).
Figure Legend Snippet: Animals with prostate cancer xenografts were randomized into control and treatment arms. Animals in treatment arm received 0.4% w/v Valproic Acid (VPA) in drinking water. Animals were treated for 35 days before excision of tumors. Tissue Microarrays (TMAs) of xenografts were created and expression of various markers was studied. Representative images of xenograft sections from control and VPA-treated groups; LNCaP, C4-2, and DU-145 (scanned at 20x magnification using the APERIO imaging system) are shown. (a) Immunohistochemistry (IHC) staining for acetylated histone 3. (b) Staining of cell cycle regulators (p21, p27, cyclin D1). (c) Staining for differentiation markers (Cytokeratin 18 (CK18), androgen receptor (AR)). (d) Staining for markers of proliferation (Ki-67), apoptosis (TUNEL), and angiogenesis (mean vascular density, MVD).

Techniques Used: Expressing, Imaging, Immunohistochemistry, Staining, TUNEL Assay

40) Product Images from "Carbon monoxide stimulates global protein methylation via its inhibitory action on cystathionine ?-synthase"

Article Title: Carbon monoxide stimulates global protein methylation via its inhibitory action on cystathionine ?-synthase

Journal: Journal of Clinical Biochemistry and Nutrition

doi: 10.3164/jcbn.11-011FR

CO alters the methylation status of histone H3. Cell lysates (25 µg protein per lane) from U937 cells were treated with CO-releasing molecule (CORM) for 4 h was separated by SDS-PAGE and performed western blot analyses with several antibodies recognized with distinct methylated residue of histone H3 described in Materials and Methods. The expression level of histone H3 protein was determined as an internal control. The data show a representative set from 3 independent experiments.
Figure Legend Snippet: CO alters the methylation status of histone H3. Cell lysates (25 µg protein per lane) from U937 cells were treated with CO-releasing molecule (CORM) for 4 h was separated by SDS-PAGE and performed western blot analyses with several antibodies recognized with distinct methylated residue of histone H3 described in Materials and Methods. The expression level of histone H3 protein was determined as an internal control. The data show a representative set from 3 independent experiments.

Techniques Used: Methylation, SDS Page, Western Blot, Expressing

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Article Snippet: .. For ChIP-seq experiments, histone H1, H3K27me3, and H3K9ac ChIPed or input DNA were used to prepare the ChIP-seq library as described (NEXTflex ChIP-seq kit, Bio Scientific, NOVA 5143-01 and NEXTflex ChIP-seq Barcodes-6, NOVA 514120) followed by high throughput sequencing with Illumina Hi-seq 2000. .. For ChIP-qPCR experiment, each sheared genomic DNA preparation was split into two equal portions and incubated with either 2 μg of MeCP2 (Diagenode, pAb-052-050) or 2 μg of histone H1 (Santa Cruz, sc-34464) or Rabbit IgG (Millipore, 12-370) as negative control.

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Article Snippet: ZW5 is the boundary-interacting protein that binds to scs, and like BEAF, ZW5 was also found in the matrix fraction, while several other nuclear proteins such as histone H3 and ABD-B were absent.

ChIP-sequencing:

Article Title: MeCP2 regulates gene expression through recognition of H3K27me3
Article Snippet: .. For ChIP-seq experiments, histone H1, H3K27me3, and H3K9ac ChIPed or input DNA were used to prepare the ChIP-seq library as described (NEXTflex ChIP-seq kit, Bio Scientific, NOVA 5143-01 and NEXTflex ChIP-seq Barcodes-6, NOVA 514120) followed by high throughput sequencing with Illumina Hi-seq 2000. .. For ChIP-qPCR experiment, each sheared genomic DNA preparation was split into two equal portions and incubated with either 2 μg of MeCP2 (Diagenode, pAb-052-050) or 2 μg of histone H1 (Santa Cruz, sc-34464) or Rabbit IgG (Millipore, 12-370) as negative control.

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    Santa Cruz Biotechnology anti phospho nfatc4
    SIRT6 overexpression suppressed the expression level and dephosphorylation of <t>NFATc4.</t> NRCMs were infected with adenovirus vector encoding SIRT6 cDNA for 48 h. (A,B) The protein expression of SIRT6 and BNP were determined by Western blot analysis. (C) The mRNA expression of NFATc4 was determined by qRT-PCR. (D) Total NFATc4 and phosphorylated NFATc4 were investigated by Western blotting. (E) The ratio of p-NFATc4/NFATc4 was determined. (F) Dual luciferase reporter assays evaluating NFATc4-dependent transcriptional activity. (G) The protein expression of calcineurin was studied in NRCMs treated with 100 μM PE for 12 h. The data were presented as mean ± SEM. ∗ P
    Anti Phospho Nfatc4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology sirt 1
    AD preconditioning improves post-ischemic recovery of ventricular function with effects partially overlapping those induced by RSV preconditioning. Hearts preconditioned with adiponectin (AD) were compared to hearts exposed to vehicle-administration (I/R) or resveratrol (RSV), used as a positive control of <t>SIRT-1</t> activation. A. Diastolic function . Left ventricular end diastolic pressure (LVEDP, mmHg) during the 180 min of reperfusion following 30 min of global ischemia (**p
    Sirt 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology sin3
    The <t>p53-Sin3</t> complex is immunologically detectable for prolonged periods following genotoxic stress, compared to the p53-p300 complex. (A) IP-Western analysis of MCF-7 cells treated with 4 Gy of irradiation (IR), 0.5 μg of adriamycin (ADR)/ml, or 4 J of UV radiation/m 2 . Cells were harvested at the indicated time points following treatment (0, 4, 8, and 24 h), and lysates were immunoprecipitated with polyclonal antisera to p53, Sin3 (AK-11; Santa Cruz Biotechnology), and p300 (Ab-1; Calbiochem), followed by Western analysis for p53 with a mouse monoclonal antibody (Ab-6; Calbiochem). Longer exposures for the p300 IPs are shown on the right, and shorter p53 exposures are shown on the left. The data depicted are representative of three independent experiments. (B) Northern analysis of the p53-repressed gene stathmin in cells treated identically to those shown in panel A. For all stresses tested, down-regulation of stathmin is evident by 8 h and increases at 24 h, concomitant with the appearance of the p53-Sin3 complex. The glyceraldehyde-3-phosphate dehydrogenase housekeeping gene (GAPDH) is included as a control for RNA loading and integrity.
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    SIRT6 overexpression suppressed the expression level and dephosphorylation of NFATc4. NRCMs were infected with adenovirus vector encoding SIRT6 cDNA for 48 h. (A,B) The protein expression of SIRT6 and BNP were determined by Western blot analysis. (C) The mRNA expression of NFATc4 was determined by qRT-PCR. (D) Total NFATc4 and phosphorylated NFATc4 were investigated by Western blotting. (E) The ratio of p-NFATc4/NFATc4 was determined. (F) Dual luciferase reporter assays evaluating NFATc4-dependent transcriptional activity. (G) The protein expression of calcineurin was studied in NRCMs treated with 100 μM PE for 12 h. The data were presented as mean ± SEM. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Suppresses NFATc4 Expression and Activation in Cardiomyocyte Hypertrophy

    doi: 10.3389/fphar.2018.01519

    Figure Lengend Snippet: SIRT6 overexpression suppressed the expression level and dephosphorylation of NFATc4. NRCMs were infected with adenovirus vector encoding SIRT6 cDNA for 48 h. (A,B) The protein expression of SIRT6 and BNP were determined by Western blot analysis. (C) The mRNA expression of NFATc4 was determined by qRT-PCR. (D) Total NFATc4 and phosphorylated NFATc4 were investigated by Western blotting. (E) The ratio of p-NFATc4/NFATc4 was determined. (F) Dual luciferase reporter assays evaluating NFATc4-dependent transcriptional activity. (G) The protein expression of calcineurin was studied in NRCMs treated with 100 μM PE for 12 h. The data were presented as mean ± SEM. ∗ P

    Article Snippet: Antibodies and Reagents Anti-NFATc4 (diluted 1:200, sc-13036), anti-phospho-NFATc4 (diluted 1:100, sc-32630) polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States).

    Techniques: Over Expression, Expressing, De-Phosphorylation Assay, Infection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay

    Changes of NFATc4 expression in pressure overload-induced cardiac hypertrophy in vivo . (A) Gross hearts from both AAC and Sham rats. Scale bar: 0.4 cm. (B) Histological analysis detected by hematoxylin and eosin staining. Scale bar: 10 μm. (C) Representative echocardiography analysis for both AAC and Sham rats. (D) The ratio of heart weight to body weight (HW/BW) were measured. Western blotting analysis was conducted to determine the protein expression of (E) BNP, (F) total NFATc4, phosphorylated NFATc4, (G) p-NFATc4/NFATc4 ratio, and (H) the protein expression of calcineurin in AAC and Sham rats. The data were presented as mean ± SEM. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Suppresses NFATc4 Expression and Activation in Cardiomyocyte Hypertrophy

    doi: 10.3389/fphar.2018.01519

    Figure Lengend Snippet: Changes of NFATc4 expression in pressure overload-induced cardiac hypertrophy in vivo . (A) Gross hearts from both AAC and Sham rats. Scale bar: 0.4 cm. (B) Histological analysis detected by hematoxylin and eosin staining. Scale bar: 10 μm. (C) Representative echocardiography analysis for both AAC and Sham rats. (D) The ratio of heart weight to body weight (HW/BW) were measured. Western blotting analysis was conducted to determine the protein expression of (E) BNP, (F) total NFATc4, phosphorylated NFATc4, (G) p-NFATc4/NFATc4 ratio, and (H) the protein expression of calcineurin in AAC and Sham rats. The data were presented as mean ± SEM. ∗ P

    Article Snippet: Antibodies and Reagents Anti-NFATc4 (diluted 1:200, sc-13036), anti-phospho-NFATc4 (diluted 1:100, sc-32630) polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States).

    Techniques: Expressing, In Vivo, Staining, Western Blot

    SIRT6 prevented PE-induced nuclear translocation of NFATc4 by interfering with calcineurin-NFATc4 interaction. NRCMs were transiently transfected with plasmid encoding the WT-SIRT6 and mutant of SIRT6 (H133Y) for 48 h followed by incubation with PE (100 μM for 12 h). (A) The protein expression of NFATc4 in the nucleus and cytoplasm was measured by Western blotting. (B) The presence of shuttling between the nucleus and cytoplasm of NFATc4 was observed by IF microscopy. The results were normalized to those of α-tubulin/LaminB1. The data were presented as mean ± SEM. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Suppresses NFATc4 Expression and Activation in Cardiomyocyte Hypertrophy

    doi: 10.3389/fphar.2018.01519

    Figure Lengend Snippet: SIRT6 prevented PE-induced nuclear translocation of NFATc4 by interfering with calcineurin-NFATc4 interaction. NRCMs were transiently transfected with plasmid encoding the WT-SIRT6 and mutant of SIRT6 (H133Y) for 48 h followed by incubation with PE (100 μM for 12 h). (A) The protein expression of NFATc4 in the nucleus and cytoplasm was measured by Western blotting. (B) The presence of shuttling between the nucleus and cytoplasm of NFATc4 was observed by IF microscopy. The results were normalized to those of α-tubulin/LaminB1. The data were presented as mean ± SEM. ∗ P

    Article Snippet: Antibodies and Reagents Anti-NFATc4 (diluted 1:200, sc-13036), anti-phospho-NFATc4 (diluted 1:100, sc-32630) polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States).

    Techniques: Translocation Assay, Transfection, Plasmid Preparation, Mutagenesis, Incubation, Expressing, Western Blot, Microscopy

    The effects of SIRT6 were dependent on deacetylase activity. NRCMs were transiently transfected with plasmid encoding the WT-SIRT6 and mutant of SIRT6 (H133Y) for 48 h followed by incubation with PE (100 μM for 12 h). (A) Western blot analysis was conducted to determine the protein expression of SIRT6. (B) The cell surface area was measured by rhodamine-phalloidin staining. Scale bar: 20 μm. (C) Western blot analysis was conducted to determine the protein expressions of BNP. (D) The mRNA expression of NFATc4 was determined by qRT-PCR. (E) The protein expression of total NFATc4, phosphorylated NFATc4 was determined by Western blotting. (F) The ratio of p-NFATc4/NFATc4 was calculated. (G) The protein expression of calcineurin was studied in NRCMs treated with 100 μM PE for 12 h. The data were presented as mean ± SEM. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Suppresses NFATc4 Expression and Activation in Cardiomyocyte Hypertrophy

    doi: 10.3389/fphar.2018.01519

    Figure Lengend Snippet: The effects of SIRT6 were dependent on deacetylase activity. NRCMs were transiently transfected with plasmid encoding the WT-SIRT6 and mutant of SIRT6 (H133Y) for 48 h followed by incubation with PE (100 μM for 12 h). (A) Western blot analysis was conducted to determine the protein expression of SIRT6. (B) The cell surface area was measured by rhodamine-phalloidin staining. Scale bar: 20 μm. (C) Western blot analysis was conducted to determine the protein expressions of BNP. (D) The mRNA expression of NFATc4 was determined by qRT-PCR. (E) The protein expression of total NFATc4, phosphorylated NFATc4 was determined by Western blotting. (F) The ratio of p-NFATc4/NFATc4 was calculated. (G) The protein expression of calcineurin was studied in NRCMs treated with 100 μM PE for 12 h. The data were presented as mean ± SEM. ∗ P

    Article Snippet: Antibodies and Reagents Anti-NFATc4 (diluted 1:200, sc-13036), anti-phospho-NFATc4 (diluted 1:100, sc-32630) polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States).

    Techniques: Histone Deacetylase Assay, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Incubation, Western Blot, Expressing, Staining, Quantitative RT-PCR

    SIRT6 interacted with NFATc4 and deacetylated NFATc4 in NRCMs. (A) The intracellular colocalization of SIRT6 (Green) and NFATc4 (Red) in NRCMs treated with 100 μM PE for 12 h. (B) Co-immunoprecipitation shows SIRT6 interact with NFATc4. (C) The acetylated level of NFATc4 was detected in NRCMs infected with or without Ad-SIRT6. (D) The intracellular colocalization of NFATc4 (Green) and p38 (Red) in NRCMs. (E) Co-immunoprecipitation shows p38 interact with NFATc4 after SIRT6 and H133Y overexpression. The data were presented as mean ± SEM. ∗∗ P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Suppresses NFATc4 Expression and Activation in Cardiomyocyte Hypertrophy

    doi: 10.3389/fphar.2018.01519

    Figure Lengend Snippet: SIRT6 interacted with NFATc4 and deacetylated NFATc4 in NRCMs. (A) The intracellular colocalization of SIRT6 (Green) and NFATc4 (Red) in NRCMs treated with 100 μM PE for 12 h. (B) Co-immunoprecipitation shows SIRT6 interact with NFATc4. (C) The acetylated level of NFATc4 was detected in NRCMs infected with or without Ad-SIRT6. (D) The intracellular colocalization of NFATc4 (Green) and p38 (Red) in NRCMs. (E) Co-immunoprecipitation shows p38 interact with NFATc4 after SIRT6 and H133Y overexpression. The data were presented as mean ± SEM. ∗∗ P

    Article Snippet: Antibodies and Reagents Anti-NFATc4 (diluted 1:200, sc-13036), anti-phospho-NFATc4 (diluted 1:100, sc-32630) polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States).

    Techniques: Immunoprecipitation, Infection, Over Expression

    Changes of NFATc4 expression in PE-induced cardiac hypertrophy in vitro . Cultured NRCMs were incubated with 100 μM PE for the indicated durations. (A) The cell surface area was measured by rhodamine-phalloidin staining. Scale bar: 20 μm. Western blotting analysis was conducted to determine the protein expression of (B) BNP, (C) total NFATc4 and phosphorylated NFATc4, (D) p-NFATc4/NFATc4 ratio, and (E) calcineurin in NRCMs treated with 100 μM PE. The data were presented as mean ± SEM. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Suppresses NFATc4 Expression and Activation in Cardiomyocyte Hypertrophy

    doi: 10.3389/fphar.2018.01519

    Figure Lengend Snippet: Changes of NFATc4 expression in PE-induced cardiac hypertrophy in vitro . Cultured NRCMs were incubated with 100 μM PE for the indicated durations. (A) The cell surface area was measured by rhodamine-phalloidin staining. Scale bar: 20 μm. Western blotting analysis was conducted to determine the protein expression of (B) BNP, (C) total NFATc4 and phosphorylated NFATc4, (D) p-NFATc4/NFATc4 ratio, and (E) calcineurin in NRCMs treated with 100 μM PE. The data were presented as mean ± SEM. ∗ P

    Article Snippet: Antibodies and Reagents Anti-NFATc4 (diluted 1:200, sc-13036), anti-phospho-NFATc4 (diluted 1:100, sc-32630) polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States).

    Techniques: Expressing, In Vitro, Cell Culture, Incubation, Staining, Western Blot

    NFATc4 participated in the anti-hypertrophic effect of SIRT6. (A–C) Western blot analysis of the protein expression of SIRT6, NFATc4, and BNP in NRCMs transfected with or without WT-NFATc4, WT-SIRT6, and treated with 100 μM PE for 12 h. (D–F) The protein expression of SIRT6, NFATc4, and BNP in NRCMs transfected with siRNAs of SIRT6, NFATc4, or negative control (NC) for 48 h. The data were presented as mean ± SEM. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Suppresses NFATc4 Expression and Activation in Cardiomyocyte Hypertrophy

    doi: 10.3389/fphar.2018.01519

    Figure Lengend Snippet: NFATc4 participated in the anti-hypertrophic effect of SIRT6. (A–C) Western blot analysis of the protein expression of SIRT6, NFATc4, and BNP in NRCMs transfected with or without WT-NFATc4, WT-SIRT6, and treated with 100 μM PE for 12 h. (D–F) The protein expression of SIRT6, NFATc4, and BNP in NRCMs transfected with siRNAs of SIRT6, NFATc4, or negative control (NC) for 48 h. The data were presented as mean ± SEM. ∗ P

    Article Snippet: Antibodies and Reagents Anti-NFATc4 (diluted 1:200, sc-13036), anti-phospho-NFATc4 (diluted 1:100, sc-32630) polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States).

    Techniques: Western Blot, Expressing, Transfection, Negative Control

    AD preconditioning improves post-ischemic recovery of ventricular function with effects partially overlapping those induced by RSV preconditioning. Hearts preconditioned with adiponectin (AD) were compared to hearts exposed to vehicle-administration (I/R) or resveratrol (RSV), used as a positive control of SIRT-1 activation. A. Diastolic function . Left ventricular end diastolic pressure (LVEDP, mmHg) during the 180 min of reperfusion following 30 min of global ischemia (**p

    Journal: PLoS ONE

    Article Title: Activation of AMPK/SIRT1 axis is required for adiponectin-mediated preconditioning on myocardial ischemia-reperfusion (I/R) injury in rats

    doi: 10.1371/journal.pone.0210654

    Figure Lengend Snippet: AD preconditioning improves post-ischemic recovery of ventricular function with effects partially overlapping those induced by RSV preconditioning. Hearts preconditioned with adiponectin (AD) were compared to hearts exposed to vehicle-administration (I/R) or resveratrol (RSV), used as a positive control of SIRT-1 activation. A. Diastolic function . Left ventricular end diastolic pressure (LVEDP, mmHg) during the 180 min of reperfusion following 30 min of global ischemia (**p

    Article Snippet: On this respect, it is important to take into account that SIRT-1 possesses at least 13 serine/threonine residues known to be target of distinct kinases (including LKB1, JNK and Cdk1), that SIRT-1 deacetylase activity increases proportionally to global phosphorylation levels, and that phosphorylation on specific sites increases the suitability of SIRT-1 as a substrate for additional kinases [ ].

    Techniques: Positive Control, Activation Assay

    AD preconditioning on isolated rat hearts protects from I/R injury via a signaling pathway involving the AMPK/LKB1/SIRT-1 axis. Phosphorylated AMPK increases eNOS-mediated NO availability, that accounts for the majority of AD cardioprotective effects. By responding to changes in ATP generation/consumption, AMPK plays a key role in modulating energy-consuming anabolic pathways and indirectly participates to SIRT-1 activation through increased NAD + levels and NAD/NADH ratio. In turn, by promoting lysine deacetylation of LKB1 and subsequent translocation from nucleus to cytoplasm, SIRT-1 may sustain the LKB1-mediated activation of AMPK. Combined treatment of AD plus CC (inhibitor of AMPK) or STN (inhibitor of SIRT-1) impair the protective effects of AD preconditioning, suggesting that both AMPK and SIRT-1 are required for AD-mediated cardiovascular protection, and regulate each other activities.

    Journal: PLoS ONE

    Article Title: Activation of AMPK/SIRT1 axis is required for adiponectin-mediated preconditioning on myocardial ischemia-reperfusion (I/R) injury in rats

    doi: 10.1371/journal.pone.0210654

    Figure Lengend Snippet: AD preconditioning on isolated rat hearts protects from I/R injury via a signaling pathway involving the AMPK/LKB1/SIRT-1 axis. Phosphorylated AMPK increases eNOS-mediated NO availability, that accounts for the majority of AD cardioprotective effects. By responding to changes in ATP generation/consumption, AMPK plays a key role in modulating energy-consuming anabolic pathways and indirectly participates to SIRT-1 activation through increased NAD + levels and NAD/NADH ratio. In turn, by promoting lysine deacetylation of LKB1 and subsequent translocation from nucleus to cytoplasm, SIRT-1 may sustain the LKB1-mediated activation of AMPK. Combined treatment of AD plus CC (inhibitor of AMPK) or STN (inhibitor of SIRT-1) impair the protective effects of AD preconditioning, suggesting that both AMPK and SIRT-1 are required for AD-mediated cardiovascular protection, and regulate each other activities.

    Article Snippet: On this respect, it is important to take into account that SIRT-1 possesses at least 13 serine/threonine residues known to be target of distinct kinases (including LKB1, JNK and Cdk1), that SIRT-1 deacetylase activity increases proportionally to global phosphorylation levels, and that phosphorylation on specific sites increases the suitability of SIRT-1 as a substrate for additional kinases [ ].

    Techniques: Isolation, Activation Assay, Translocation Assay

    Inhibition of either AMPK or SIRT-1 activity abrogates AD-mediated preconditioning on infarct mass extension. A. Upper panel. Quantification of necrotic tissue, expressed as percentage of the left ventricular mass, in hearts exposed to various treatments at the end of the reperfusion interval. Statistical differences between groups were evaluated by one-factor ANOVA followed by Bonferroni correction. Values are expressed as mean ± SEM (**p

    Journal: PLoS ONE

    Article Title: Activation of AMPK/SIRT1 axis is required for adiponectin-mediated preconditioning on myocardial ischemia-reperfusion (I/R) injury in rats

    doi: 10.1371/journal.pone.0210654

    Figure Lengend Snippet: Inhibition of either AMPK or SIRT-1 activity abrogates AD-mediated preconditioning on infarct mass extension. A. Upper panel. Quantification of necrotic tissue, expressed as percentage of the left ventricular mass, in hearts exposed to various treatments at the end of the reperfusion interval. Statistical differences between groups were evaluated by one-factor ANOVA followed by Bonferroni correction. Values are expressed as mean ± SEM (**p

    Article Snippet: On this respect, it is important to take into account that SIRT-1 possesses at least 13 serine/threonine residues known to be target of distinct kinases (including LKB1, JNK and Cdk1), that SIRT-1 deacetylase activity increases proportionally to global phosphorylation levels, and that phosphorylation on specific sites increases the suitability of SIRT-1 as a substrate for additional kinases [ ].

    Techniques: Inhibition, Activity Assay

    The AD-mediated post-ischemic left ventricular recovery partially depends on SIRT-1. Hearts preconditioned with AD were compared to hearts infused with sirtinol, a specific inhibitor of SIRT-1, alone (STN), or in combination with AD (AD + STN). A . Diastolic function . Left ventricular end diastolic pressure (LVEDP, mmHg) during the 180 min of reperfusion (**p

    Journal: PLoS ONE

    Article Title: Activation of AMPK/SIRT1 axis is required for adiponectin-mediated preconditioning on myocardial ischemia-reperfusion (I/R) injury in rats

    doi: 10.1371/journal.pone.0210654

    Figure Lengend Snippet: The AD-mediated post-ischemic left ventricular recovery partially depends on SIRT-1. Hearts preconditioned with AD were compared to hearts infused with sirtinol, a specific inhibitor of SIRT-1, alone (STN), or in combination with AD (AD + STN). A . Diastolic function . Left ventricular end diastolic pressure (LVEDP, mmHg) during the 180 min of reperfusion (**p

    Article Snippet: On this respect, it is important to take into account that SIRT-1 possesses at least 13 serine/threonine residues known to be target of distinct kinases (including LKB1, JNK and Cdk1), that SIRT-1 deacetylase activity increases proportionally to global phosphorylation levels, and that phosphorylation on specific sites increases the suitability of SIRT-1 as a substrate for additional kinases [ ].

    Techniques:

    AD-mediated preconditioning activates AMPK, LKB1, and eNOS signaling pathways in the early post-ischemic reperfusion (15 min). Lysates from hearts exposed to 15 min of reperfusion were immunoblotted for phosphorylated and total isoforms of the indicated proteins. A. For each experimental condition, the ratio of phosphorylated/total SIRT-1, eNOS, AMPK and LKB1 proteins are expressed as the mean ± SEM of at least 3 independent experiments (each run in triplicate). Statistical differences between groups were evaluated by one-factor ANOVA followed by Bonferroni correction (*p

    Journal: PLoS ONE

    Article Title: Activation of AMPK/SIRT1 axis is required for adiponectin-mediated preconditioning on myocardial ischemia-reperfusion (I/R) injury in rats

    doi: 10.1371/journal.pone.0210654

    Figure Lengend Snippet: AD-mediated preconditioning activates AMPK, LKB1, and eNOS signaling pathways in the early post-ischemic reperfusion (15 min). Lysates from hearts exposed to 15 min of reperfusion were immunoblotted for phosphorylated and total isoforms of the indicated proteins. A. For each experimental condition, the ratio of phosphorylated/total SIRT-1, eNOS, AMPK and LKB1 proteins are expressed as the mean ± SEM of at least 3 independent experiments (each run in triplicate). Statistical differences between groups were evaluated by one-factor ANOVA followed by Bonferroni correction (*p

    Article Snippet: On this respect, it is important to take into account that SIRT-1 possesses at least 13 serine/threonine residues known to be target of distinct kinases (including LKB1, JNK and Cdk1), that SIRT-1 deacetylase activity increases proportionally to global phosphorylation levels, and that phosphorylation on specific sites increases the suitability of SIRT-1 as a substrate for additional kinases [ ].

    Techniques:

    HDAC6 deficiency results in accumulation of SOD1 G93A mutant protein aggregates in the spinal cord (A) 129/Black Swiss/B6SJL background mice for each genotype were sacrificed at 20 weeks of age, and dissected spinal cord were subjected to the immunostaining with anti-ubiquitin antibody for ubiquitinated protein aggregates. Arrowheads indicate ubiquitinated protein aggregates. (B) Spinal cord lysates (100 ug) from indicated genotypes at 20 weeks old were subjected to filter trap assay to measure protein aggregates, using anti-ubiquitin (bottom panel) and anti-human SOD1 antibody (top panel). (C) The same spinal cord lysates were subjected to immunoblotting using anti-human SOD1, anti-HDAC6, anti-acetyl tubulin and anti-actin antibodies.

    Journal: Neuro-degenerative diseases

    Article Title: Uncoupling of protein aggregation and neurodegeneration in a mouse ALS model

    doi: 10.1159/000437208

    Figure Lengend Snippet: HDAC6 deficiency results in accumulation of SOD1 G93A mutant protein aggregates in the spinal cord (A) 129/Black Swiss/B6SJL background mice for each genotype were sacrificed at 20 weeks of age, and dissected spinal cord were subjected to the immunostaining with anti-ubiquitin antibody for ubiquitinated protein aggregates. Arrowheads indicate ubiquitinated protein aggregates. (B) Spinal cord lysates (100 ug) from indicated genotypes at 20 weeks old were subjected to filter trap assay to measure protein aggregates, using anti-ubiquitin (bottom panel) and anti-human SOD1 antibody (top panel). (C) The same spinal cord lysates were subjected to immunoblotting using anti-human SOD1, anti-HDAC6, anti-acetyl tubulin and anti-actin antibodies.

    Article Snippet: HDAC6 is a microtubule-associated deacetylase.

    Techniques: Mutagenesis, Mouse Assay, Immunostaining, TRAP Assay

    HDAC6 is required for efficient targeting of lysosomes to aggregates (A) Wild type, HDAC6 KO and HDAC6 KO MEFs that stably express human HDAC6WT, HDAC6CD or HDAC6ΔBuz were treated with MG132 (2.5 μM for 24 hours) and changed with normal growth media for 18 hours to allow the degradation of non-aggregated protein. Cells were immunostained with anti-LAMP-1 antibody for lysosomes and anti-ubiquitin antibody for aggregates. Open arrows indicated ubiquitin-positive aggregates that contain LAMP-1. (B) Quantification of lysosomes targeted to ubiquitin-positive aggregates. The graph represents the percent of LAMP-1-positive aggregates. The mean of LAMP-1-positive aggregates from 3 independent experiments (more than 10 images per each experiment) are presented with standard deviation for error bar. (t-test, **p

    Journal: Neuro-degenerative diseases

    Article Title: Uncoupling of protein aggregation and neurodegeneration in a mouse ALS model

    doi: 10.1159/000437208

    Figure Lengend Snippet: HDAC6 is required for efficient targeting of lysosomes to aggregates (A) Wild type, HDAC6 KO and HDAC6 KO MEFs that stably express human HDAC6WT, HDAC6CD or HDAC6ΔBuz were treated with MG132 (2.5 μM for 24 hours) and changed with normal growth media for 18 hours to allow the degradation of non-aggregated protein. Cells were immunostained with anti-LAMP-1 antibody for lysosomes and anti-ubiquitin antibody for aggregates. Open arrows indicated ubiquitin-positive aggregates that contain LAMP-1. (B) Quantification of lysosomes targeted to ubiquitin-positive aggregates. The graph represents the percent of LAMP-1-positive aggregates. The mean of LAMP-1-positive aggregates from 3 independent experiments (more than 10 images per each experiment) are presented with standard deviation for error bar. (t-test, **p

    Article Snippet: HDAC6 is a microtubule-associated deacetylase.

    Techniques: Stable Transfection, Standard Deviation, T-Test

    HDAC6-knockdown cells are sensitive to misfolded proteins associated with neurodegeneration (Ai) Control and HDAC6-knockdown 293T cells were transfected with GFP-fused cytosolic (GFP-CFTR ΔF508 and GFP-AR-112Q) or nuclear aggregates (GFP-Ataxin-1 82Q) and fractionated into SDS soluble (S) and SDS insoluble fractions (I). Each fractionated sample was subjected for immunoblot analysis using anti-GFP, anti-HDAC6 and anti-tubulin antibodies. (Aii) Western blotting images were analyzed by BIO-1D™ software and relative band intensity of SDS insoluble aggregates were presented in bottom panel graph. (B) Control and HDAC6-knockdown A549 cells were transfected with cytosolic (CFTR ΔF508, AR112Q, Htt 81Q, SOD1 G93A ) or nuclear protein aggregates (ataxin-1) expression plasmids. Cells were immunostained with anti-active-caspase-3 antibody to check apoptotic cells and the average of apoptotic cells percent from three independent experiments are presented with standard deviation as error bar (t-test, *: p

    Journal: Neuro-degenerative diseases

    Article Title: Uncoupling of protein aggregation and neurodegeneration in a mouse ALS model

    doi: 10.1159/000437208

    Figure Lengend Snippet: HDAC6-knockdown cells are sensitive to misfolded proteins associated with neurodegeneration (Ai) Control and HDAC6-knockdown 293T cells were transfected with GFP-fused cytosolic (GFP-CFTR ΔF508 and GFP-AR-112Q) or nuclear aggregates (GFP-Ataxin-1 82Q) and fractionated into SDS soluble (S) and SDS insoluble fractions (I). Each fractionated sample was subjected for immunoblot analysis using anti-GFP, anti-HDAC6 and anti-tubulin antibodies. (Aii) Western blotting images were analyzed by BIO-1D™ software and relative band intensity of SDS insoluble aggregates were presented in bottom panel graph. (B) Control and HDAC6-knockdown A549 cells were transfected with cytosolic (CFTR ΔF508, AR112Q, Htt 81Q, SOD1 G93A ) or nuclear protein aggregates (ataxin-1) expression plasmids. Cells were immunostained with anti-active-caspase-3 antibody to check apoptotic cells and the average of apoptotic cells percent from three independent experiments are presented with standard deviation as error bar (t-test, *: p

    Article Snippet: HDAC6 is a microtubule-associated deacetylase.

    Techniques: Transfection, Western Blot, Software, Expressing, Standard Deviation, T-Test

    HDAC6 deficiency moderately accelerates the pathogenesis of ALS disease in mouse model (A) Wild type and HDAC6 KO mice with 129/Black Swiss with double genetic background were subjected to accelerating rotarod test (4-40rpm/300 sec) at 6 month old. Average of latency to fall are presented with standard error as error bar. (B) 129/Black Swiss/B6SJL triple genetic background mice with indicated genotypes were subjected to accelerating rotarod test (4-40rpm/180 sec) at every week from 9 weeks to 23 weeks old. Latency to fall was recorded as highest score of three trials and presented as average of group. Cut-off is 180 seconds. Average of latency to fall were presented with standard deviation as error bar. (C) The mouse spinal cord sections are stained with anti-HB-9 antibody to visualize motor neuron for counting at 20 weeks old. Quantification of motor neuron in spinal cords from indicated genotype mouse are presented with standard deviation for error bar. (t-test, **: p

    Journal: Neuro-degenerative diseases

    Article Title: Uncoupling of protein aggregation and neurodegeneration in a mouse ALS model

    doi: 10.1159/000437208

    Figure Lengend Snippet: HDAC6 deficiency moderately accelerates the pathogenesis of ALS disease in mouse model (A) Wild type and HDAC6 KO mice with 129/Black Swiss with double genetic background were subjected to accelerating rotarod test (4-40rpm/300 sec) at 6 month old. Average of latency to fall are presented with standard error as error bar. (B) 129/Black Swiss/B6SJL triple genetic background mice with indicated genotypes were subjected to accelerating rotarod test (4-40rpm/180 sec) at every week from 9 weeks to 23 weeks old. Latency to fall was recorded as highest score of three trials and presented as average of group. Cut-off is 180 seconds. Average of latency to fall were presented with standard deviation as error bar. (C) The mouse spinal cord sections are stained with anti-HB-9 antibody to visualize motor neuron for counting at 20 weeks old. Quantification of motor neuron in spinal cords from indicated genotype mouse are presented with standard deviation for error bar. (t-test, **: p

    Article Snippet: HDAC6 is a microtubule-associated deacetylase.

    Techniques: Mouse Assay, Size-exclusion Chromatography, Standard Deviation, Staining, T-Test

    The p53-Sin3 complex is immunologically detectable for prolonged periods following genotoxic stress, compared to the p53-p300 complex. (A) IP-Western analysis of MCF-7 cells treated with 4 Gy of irradiation (IR), 0.5 μg of adriamycin (ADR)/ml, or 4 J of UV radiation/m 2 . Cells were harvested at the indicated time points following treatment (0, 4, 8, and 24 h), and lysates were immunoprecipitated with polyclonal antisera to p53, Sin3 (AK-11; Santa Cruz Biotechnology), and p300 (Ab-1; Calbiochem), followed by Western analysis for p53 with a mouse monoclonal antibody (Ab-6; Calbiochem). Longer exposures for the p300 IPs are shown on the right, and shorter p53 exposures are shown on the left. The data depicted are representative of three independent experiments. (B) Northern analysis of the p53-repressed gene stathmin in cells treated identically to those shown in panel A. For all stresses tested, down-regulation of stathmin is evident by 8 h and increases at 24 h, concomitant with the appearance of the p53-Sin3 complex. The glyceraldehyde-3-phosphate dehydrogenase housekeeping gene (GAPDH) is included as a control for RNA loading and integrity.

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: The p53-Sin3 complex is immunologically detectable for prolonged periods following genotoxic stress, compared to the p53-p300 complex. (A) IP-Western analysis of MCF-7 cells treated with 4 Gy of irradiation (IR), 0.5 μg of adriamycin (ADR)/ml, or 4 J of UV radiation/m 2 . Cells were harvested at the indicated time points following treatment (0, 4, 8, and 24 h), and lysates were immunoprecipitated with polyclonal antisera to p53, Sin3 (AK-11; Santa Cruz Biotechnology), and p300 (Ab-1; Calbiochem), followed by Western analysis for p53 with a mouse monoclonal antibody (Ab-6; Calbiochem). Longer exposures for the p300 IPs are shown on the right, and shorter p53 exposures are shown on the left. The data depicted are representative of three independent experiments. (B) Northern analysis of the p53-repressed gene stathmin in cells treated identically to those shown in panel A. For all stresses tested, down-regulation of stathmin is evident by 8 h and increases at 24 h, concomitant with the appearance of the p53-Sin3 complex. The glyceraldehyde-3-phosphate dehydrogenase housekeeping gene (GAPDH) is included as a control for RNA loading and integrity.

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Western Blot, Irradiation, Immunoprecipitation, Northern Blot

    Model for the differential stability of p53-dependent transactivation and transrepression complexes. p53-dependent transactivation of p53-induced genes such as p21 utilizes p300 as a coactivator; p300 serves as a scaffold for interaction between p53 and MDM2, thus targeting p53 for degradation and prohibiting prolonged transactivation. In contrast, the p53 protein present at the promoters of p53-repressed genes like map4 would be bound to the corepressor Sin3 (shown here bound to histone deacetylase 1 [HDAC-1]) and is predicted to be protected from proteasome-mediated degradation. Such protection would facilitate prolonged and efficient transcriptional repression.

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: Model for the differential stability of p53-dependent transactivation and transrepression complexes. p53-dependent transactivation of p53-induced genes such as p21 utilizes p300 as a coactivator; p300 serves as a scaffold for interaction between p53 and MDM2, thus targeting p53 for degradation and prohibiting prolonged transactivation. In contrast, the p53 protein present at the promoters of p53-repressed genes like map4 would be bound to the corepressor Sin3 (shown here bound to histone deacetylase 1 [HDAC-1]) and is predicted to be protected from proteasome-mediated degradation. Such protection would facilitate prolonged and efficient transcriptional repression.

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Histone Deacetylase Assay

    Stabilization of p53 by Sin3 occurs in an MDM2-independent manner. (A) Western analysis of 174-1 cells (p53 −/− MDM2 −/− transfected with wt p53 in the presence of parental vector alone (lane 1), Sin3 expression construct (lane 2), or p14 ARF (lane 3). (B) Western analysis of H1299 cells transfected with wt p53 (lanes 1 to 3) or the tumor-derived mutant R175Y (lanes 4 to 6) in the presence of vector alone (lanes 1 and 4), Sin3 expression construct (lanes 2 and 5), and p14 ARF (lanes 3 and 6). Equal protein loading among the lanes was confirmed by Western analysis for β-actin.

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: Stabilization of p53 by Sin3 occurs in an MDM2-independent manner. (A) Western analysis of 174-1 cells (p53 −/− MDM2 −/− transfected with wt p53 in the presence of parental vector alone (lane 1), Sin3 expression construct (lane 2), or p14 ARF (lane 3). (B) Western analysis of H1299 cells transfected with wt p53 (lanes 1 to 3) or the tumor-derived mutant R175Y (lanes 4 to 6) in the presence of vector alone (lanes 1 and 4), Sin3 expression construct (lanes 2 and 5), and p14 ARF (lanes 3 and 6). Equal protein loading among the lanes was confirmed by Western analysis for β-actin.

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Construct, Derivative Assay, Mutagenesis

    Sin3 stabilizes p53 and protects it from MDM2-mediated degradation. (A) Western analysis of p53 (Ab-6; Calbiochem) in lysates made from H1299 cells transiently transfected with wt p53 in the presence of parental vector alone (pRc/CMV) (lane 1), pCMX-Sin3 (lane 2), or p14 ARF (lane 3). Equivalent protein loading between lanes was confirmed by Western analysis of β-actin levels. (B) Western analysis of MDM2 (Ab-1; Calbiochem) in H1299 cells transfected with parental vector alone (pRc/CMV) (lane 1) or a cytomegalovirus (CMV)-driven MDM2 expression construct, in the presence (lane 3) and absence (lane 2) of an equal amount, in micrograms, of cotransfected Sin3. Equivalent protein loading was confirmed by Western analysis of β-actin. (C) Western analysis of MDM2 and p53 in H1299 cells transfected with wt p53 in the presence of MDM2 (lane 2) or MDM2 and Sin3 (lane 3). While transfection with MDM2 leads to significant decreases in p53 steady-state levels (lane 2), this effect is reversed by transfection with Sin3 (lane 3). Equal levels of protein loading were confirmed by Western analysis of β-actin levels; the occasional differences in p53 levels relative to β-actin represent the use of different p53 antisera (Ab-6 or p53 fl1-393 [Santa Cruz Biotechnology]) as well as different actin antisera (AC-15 [Sigma] versus antiactin polyclonal Santa Cruz [Biotechnology]). While these antisera occasionally revealed different levels of p53 and/or actin, the trends in p53 stabilization in the presence of Sin3 were consistent.

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: Sin3 stabilizes p53 and protects it from MDM2-mediated degradation. (A) Western analysis of p53 (Ab-6; Calbiochem) in lysates made from H1299 cells transiently transfected with wt p53 in the presence of parental vector alone (pRc/CMV) (lane 1), pCMX-Sin3 (lane 2), or p14 ARF (lane 3). Equivalent protein loading between lanes was confirmed by Western analysis of β-actin levels. (B) Western analysis of MDM2 (Ab-1; Calbiochem) in H1299 cells transfected with parental vector alone (pRc/CMV) (lane 1) or a cytomegalovirus (CMV)-driven MDM2 expression construct, in the presence (lane 3) and absence (lane 2) of an equal amount, in micrograms, of cotransfected Sin3. Equivalent protein loading was confirmed by Western analysis of β-actin. (C) Western analysis of MDM2 and p53 in H1299 cells transfected with wt p53 in the presence of MDM2 (lane 2) or MDM2 and Sin3 (lane 3). While transfection with MDM2 leads to significant decreases in p53 steady-state levels (lane 2), this effect is reversed by transfection with Sin3 (lane 3). Equal levels of protein loading were confirmed by Western analysis of β-actin levels; the occasional differences in p53 levels relative to β-actin represent the use of different p53 antisera (Ab-6 or p53 fl1-393 [Santa Cruz Biotechnology]) as well as different actin antisera (AC-15 [Sigma] versus antiactin polyclonal Santa Cruz [Biotechnology]). While these antisera occasionally revealed different levels of p53 and/or actin, the trends in p53 stabilization in the presence of Sin3 were consistent.

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Construct

    Sin3 inhibits proteasome-mediated degradation of p53. (A) Western analysis of p53 levels in H1299 cells transfected with wt p53 in the presence of cotransfected Sin3 (lanes 2 and 5) or p14 ARF (lanes 3 and 6). Lanes 4 to 6 include a 2-h posttransfection incubation with a 200 μM concentration of the proteasome inhibitor MG132, while lanes 1 to 3 are treated with dilution vehicle (DMSO). While MG132 is able to stabilize transfected p53 approximately threefold (compare lanes 1 and 4), it has an insignificant effect on the level of p53 stabilized by Sin3 (compare lanes 2 and 5), indicating that Sin3 and MG132 likely function through redundant pathways (inhibition of proteasome-mediated degradation). (B) The calpain 1 inhibitor ALLN is able to further stabilize Sin3-stabilized p53 (compare lanes 2 and 4). Both Sin3 transfection and ALLN treatment (45 μM) result in fivefold increases in p53 levels; the two agents together function additively (lane 4). Equal protein loading was confirmed by Western analysis for β-actin.

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: Sin3 inhibits proteasome-mediated degradation of p53. (A) Western analysis of p53 levels in H1299 cells transfected with wt p53 in the presence of cotransfected Sin3 (lanes 2 and 5) or p14 ARF (lanes 3 and 6). Lanes 4 to 6 include a 2-h posttransfection incubation with a 200 μM concentration of the proteasome inhibitor MG132, while lanes 1 to 3 are treated with dilution vehicle (DMSO). While MG132 is able to stabilize transfected p53 approximately threefold (compare lanes 1 and 4), it has an insignificant effect on the level of p53 stabilized by Sin3 (compare lanes 2 and 5), indicating that Sin3 and MG132 likely function through redundant pathways (inhibition of proteasome-mediated degradation). (B) The calpain 1 inhibitor ALLN is able to further stabilize Sin3-stabilized p53 (compare lanes 2 and 4). Both Sin3 transfection and ALLN treatment (45 μM) result in fivefold increases in p53 levels; the two agents together function additively (lane 4). Equal protein loading was confirmed by Western analysis for β-actin.

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Western Blot, Transfection, Incubation, Concentration Assay, Inhibition

    Stabilization of p53 by Sin3 requires the Sin3-binding domain of p53 (amino acids 61 to 75). (A) Western analysis of H1299 cells transiently transfected with wt p53 (lanes 1 and 2), the p53TZ construct encoding wt p53 but containing an artificial TZ (lanes 3 and 4), and a deletion mutant of this protein that lacks amino acids 1 to 100 and fails to interact with Sin3 (Δ1–100TZ) (lanes 5 and 6). Odd-numbered lanes are cotransfected with parental vector, and even-numbered lanes are cotransfected with an equal amount of Sin3 expression construct. (B) Western analysis of a p53 deletion mutant with amino acids 44 to 61 deleted (Δ44–61TZ). This mutant is stabilized by exogenous Sin3 (lanes 1 and 2) and binds to Sin3 in transfected H1299 cells that are immunoprecipitated with antiserum to Sin3 (AK-11) and immunoblotted with p53 antiserum (Ab-6; Calbiochem) (lanes 3 and 4). (C) Western analysis of H1299 cells transfected with the p53TZ cDNA or with internal deletions created in the p53TZ backbone (Δ61–75TZ) (lanes 3 and 4) and Δ81–96TZ (lanes 5 and 6). Odd-numbered lanes are cotransfected with parental vector, and even-numbered lanes are cotransfected with Sin3 expression construct. Only the Δ61–75 mutant fails to be stabilized by exogenous Sin3 (lane 4). (D) IP-Western analysis of the interaction of Sin3 in vivo with deletion mutants of p53, including the p53 mutant that fails to be stabilized by Sin3 (Δ61–75TZ) (lanes 3 and 4). Odd-numbered lanes are cotransfected with parental vector, and even-numbered lanes are cotransfected with Sin3 expression construct.

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: Stabilization of p53 by Sin3 requires the Sin3-binding domain of p53 (amino acids 61 to 75). (A) Western analysis of H1299 cells transiently transfected with wt p53 (lanes 1 and 2), the p53TZ construct encoding wt p53 but containing an artificial TZ (lanes 3 and 4), and a deletion mutant of this protein that lacks amino acids 1 to 100 and fails to interact with Sin3 (Δ1–100TZ) (lanes 5 and 6). Odd-numbered lanes are cotransfected with parental vector, and even-numbered lanes are cotransfected with an equal amount of Sin3 expression construct. (B) Western analysis of a p53 deletion mutant with amino acids 44 to 61 deleted (Δ44–61TZ). This mutant is stabilized by exogenous Sin3 (lanes 1 and 2) and binds to Sin3 in transfected H1299 cells that are immunoprecipitated with antiserum to Sin3 (AK-11) and immunoblotted with p53 antiserum (Ab-6; Calbiochem) (lanes 3 and 4). (C) Western analysis of H1299 cells transfected with the p53TZ cDNA or with internal deletions created in the p53TZ backbone (Δ61–75TZ) (lanes 3 and 4) and Δ81–96TZ (lanes 5 and 6). Odd-numbered lanes are cotransfected with parental vector, and even-numbered lanes are cotransfected with Sin3 expression construct. Only the Δ61–75 mutant fails to be stabilized by exogenous Sin3 (lane 4). (D) IP-Western analysis of the interaction of Sin3 in vivo with deletion mutants of p53, including the p53 mutant that fails to be stabilized by Sin3 (Δ61–75TZ) (lanes 3 and 4). Odd-numbered lanes are cotransfected with parental vector, and even-numbered lanes are cotransfected with Sin3 expression construct.

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Binding Assay, Western Blot, Transfection, Construct, Mutagenesis, Plasmid Preparation, Expressing, Immunoprecipitation, In Vivo

    A p53 deletion mutant lacking the Sin3-binding domain (Δ61–75) shows enhanced stability in vivo. (A) Pulse-chase analysis of 35 S-methionine radiolabeled H1299 cells transfected with wt p53 or the p53 mutant lacking the Sin3-binding domain (Δ61–75). Cells were radiolabeled with [ 35 S]methionine and chased for the indicated time points with excess unlabeled methionine. Equal counts per minute of lysate were immunoprecipitated with polyclonal antisera to p53 (Santa Cruz Biotechnology), as indicated in Materials and Methods. The asterisk (*) indicates β-actin, which is frequently nonspecifically immunoprecipitated by polyclonal antisera to p53. The graph depicts densitometric analysis of this representative experiment, which indicates that wt p53 has a 2-h half-life, while the Δ61–75 mutant has an approximately 6-h half-life. (B) Western analysis of the half-life of wt p53 (lanes 1 to 4) and the Δ61–75 mutant (lanes 5 to 8) following transfection of H1299 cells for 24 h and treatment with 40 μg of cycloheximide/ml for the times indicated to halt new protein synthesis. The data shown are representative of at least three independent experiments; β-actin is shown to control for protein loading. (C) Northern analysis of H1299 cells transiently transfected with 0, 1, and 5 μg of the wt p53TZ cDNA or the Δ61–75 deletion mutant (Δ61–75TZ). Thirty-six hours following transfection, cells were harvested and analyzed by Northern analysis for the level of endogenous p21 , which was up-regulated equally well by both forms of p53. A picture of the ethidium bromide-stained gel used as a control for RNA loading and integrity is included. (D) Western analysis of p53 levels in cells transfected with parental vector alone (odd-numbered lanes) or vector expressing MDM2 (even-numbered lanes). Both wt p53 (lanes 1 and 2) and the Δ61–75 mutant (lanes 3 and 4) are effectively degraded by MDM2.

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: A p53 deletion mutant lacking the Sin3-binding domain (Δ61–75) shows enhanced stability in vivo. (A) Pulse-chase analysis of 35 S-methionine radiolabeled H1299 cells transfected with wt p53 or the p53 mutant lacking the Sin3-binding domain (Δ61–75). Cells were radiolabeled with [ 35 S]methionine and chased for the indicated time points with excess unlabeled methionine. Equal counts per minute of lysate were immunoprecipitated with polyclonal antisera to p53 (Santa Cruz Biotechnology), as indicated in Materials and Methods. The asterisk (*) indicates β-actin, which is frequently nonspecifically immunoprecipitated by polyclonal antisera to p53. The graph depicts densitometric analysis of this representative experiment, which indicates that wt p53 has a 2-h half-life, while the Δ61–75 mutant has an approximately 6-h half-life. (B) Western analysis of the half-life of wt p53 (lanes 1 to 4) and the Δ61–75 mutant (lanes 5 to 8) following transfection of H1299 cells for 24 h and treatment with 40 μg of cycloheximide/ml for the times indicated to halt new protein synthesis. The data shown are representative of at least three independent experiments; β-actin is shown to control for protein loading. (C) Northern analysis of H1299 cells transiently transfected with 0, 1, and 5 μg of the wt p53TZ cDNA or the Δ61–75 deletion mutant (Δ61–75TZ). Thirty-six hours following transfection, cells were harvested and analyzed by Northern analysis for the level of endogenous p21 , which was up-regulated equally well by both forms of p53. A picture of the ethidium bromide-stained gel used as a control for RNA loading and integrity is included. (D) Western analysis of p53 levels in cells transfected with parental vector alone (odd-numbered lanes) or vector expressing MDM2 (even-numbered lanes). Both wt p53 (lanes 1 and 2) and the Δ61–75 mutant (lanes 3 and 4) are effectively degraded by MDM2.

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Mutagenesis, Binding Assay, In Vivo, Pulse Chase, Transfection, Immunoprecipitation, Western Blot, Northern Blot, Staining, Plasmid Preparation, Expressing

    Sin3 stabilizes endogenous p53 in MCF-7 cells. (A) Immunofluorescence of MCF-7 cells transfected with a GFP expression construct (pEGFP), analyzed by differential interference contrast (A1 and C1), for GFP fluorescence (A2 and C2) and for p53 staining (A3 and C3) with polyclonal antisera to p53. GFP-positive cells were scored as transfected cells, and cells with high levels of p53 immunostaining were scored as positive for p53. In three independent blinded studies, over 100 GFP-positive cells were scored for strong p53 immunostaining; the combined results from these experiments are presented in panel B. (B) The averaged data from three independent experiments indicate that transfection with Sin3 and p14 ARF leads to significant increases in the number of transfected cells with strong immunopositivity for p53. While 27% of cells transfected with GFP alone show strong immunostaining for p53, 65 and 74% of cells transfected with Sin3 and p14 ARF , respectively, showed increased p53 immunostaining. The asterisks indicate a statistically significant difference between the indicated treatment and control ( P

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: Sin3 stabilizes endogenous p53 in MCF-7 cells. (A) Immunofluorescence of MCF-7 cells transfected with a GFP expression construct (pEGFP), analyzed by differential interference contrast (A1 and C1), for GFP fluorescence (A2 and C2) and for p53 staining (A3 and C3) with polyclonal antisera to p53. GFP-positive cells were scored as transfected cells, and cells with high levels of p53 immunostaining were scored as positive for p53. In three independent blinded studies, over 100 GFP-positive cells were scored for strong p53 immunostaining; the combined results from these experiments are presented in panel B. (B) The averaged data from three independent experiments indicate that transfection with Sin3 and p14 ARF leads to significant increases in the number of transfected cells with strong immunopositivity for p53. While 27% of cells transfected with GFP alone show strong immunostaining for p53, 65 and 74% of cells transfected with Sin3 and p14 ARF , respectively, showed increased p53 immunostaining. The asterisks indicate a statistically significant difference between the indicated treatment and control ( P

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Immunofluorescence, Transfection, Expressing, Construct, Fluorescence, Staining, Immunostaining

    A point mutant of p53 containing arginine instead of proline at amino acid 71 (P71R) fails to interact with Sin3 in vivo or to be stabilized by exogenous Sin3. This mutant shows transactivation potential, however, that is indistinguishable from wt p53. (A) Transfection of H1299 cells with wt p53 or point mutants containing leucine at amino acid 80 (P80L) or arginine at amino acid 71 (P71R) followed by IP with Sin3 antiserum (AK-11; Santa Cruz Biotechnology) and immunoblotting with p53 antiserum (Ab-6; Calbiochem). The data indicate that the P71R mutant shows impaired interaction with Sin3 in transfected cells. (B) Transfection of H1299 cells with 50 ng of p53 or the P71R mutant, along with increasing concentrations of Sin3 expression plasmid (0.5 and 2.5 μg, lanes 2, 3, 5, and 6). Immunoblot analysis of p53 levels indicates that p53 is stabilized by Sin3 in a dose-dependent manner but that neither dose of Sin3 is capable of stabilizing the P71R mutant, which shows a defective interaction with Sin3. A β-actin control is included for protein loading. (C) Luciferase activity of the p53-inducible luciferase vector SpVII (a minimal promoter containing the E1B TATA box and a single p53 consensus element) transiently transfected into H1299 cells in the presence of parental vector (pRc/CMV) or increasing concentrations of wt p53 (25 and 100 ng) or the P71R point mutant of p53. The data depicted represent the fold increase in luciferase activity obtained after transfection, averaged from five independent experiments. Error bars depict the standard error of the mean for the five experiments. (D) Western analysis depicting comparable p53 levels expressed in the transfected cells analyzed in the results shown in panel C. A total of 1.25 μg of the SpVII luciferase reporter construct was cotransfected with either vector alone or increasing concentrations of wt p53 (25 and 100 ng), P71R (25 and 100 ng), and 100 ng of pEGFP (transfection control) in six-well plates and analyzed for p53 levels by Western analysis. Western analysis of the GFP levels between samples is used to show relatively equal transfection efficiencies, and equivalent protein loading is depicted by Western analysis for β-actin.

    Journal: Molecular and Cellular Biology

    Article Title: The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.12.3974-3985.2001

    Figure Lengend Snippet: A point mutant of p53 containing arginine instead of proline at amino acid 71 (P71R) fails to interact with Sin3 in vivo or to be stabilized by exogenous Sin3. This mutant shows transactivation potential, however, that is indistinguishable from wt p53. (A) Transfection of H1299 cells with wt p53 or point mutants containing leucine at amino acid 80 (P80L) or arginine at amino acid 71 (P71R) followed by IP with Sin3 antiserum (AK-11; Santa Cruz Biotechnology) and immunoblotting with p53 antiserum (Ab-6; Calbiochem). The data indicate that the P71R mutant shows impaired interaction with Sin3 in transfected cells. (B) Transfection of H1299 cells with 50 ng of p53 or the P71R mutant, along with increasing concentrations of Sin3 expression plasmid (0.5 and 2.5 μg, lanes 2, 3, 5, and 6). Immunoblot analysis of p53 levels indicates that p53 is stabilized by Sin3 in a dose-dependent manner but that neither dose of Sin3 is capable of stabilizing the P71R mutant, which shows a defective interaction with Sin3. A β-actin control is included for protein loading. (C) Luciferase activity of the p53-inducible luciferase vector SpVII (a minimal promoter containing the E1B TATA box and a single p53 consensus element) transiently transfected into H1299 cells in the presence of parental vector (pRc/CMV) or increasing concentrations of wt p53 (25 and 100 ng) or the P71R point mutant of p53. The data depicted represent the fold increase in luciferase activity obtained after transfection, averaged from five independent experiments. Error bars depict the standard error of the mean for the five experiments. (D) Western analysis depicting comparable p53 levels expressed in the transfected cells analyzed in the results shown in panel C. A total of 1.25 μg of the SpVII luciferase reporter construct was cotransfected with either vector alone or increasing concentrations of wt p53 (25 and 100 ng), P71R (25 and 100 ng), and 100 ng of pEGFP (transfection control) in six-well plates and analyzed for p53 levels by Western analysis. Western analysis of the GFP levels between samples is used to show relatively equal transfection efficiencies, and equivalent protein loading is depicted by Western analysis for β-actin.

    Article Snippet: Western analysis of transfected cells revealed that wt p53 was expressed at four- to fivefold-higher levels when cells were transfected with Sin3 (Fig. A, lane 2).

    Techniques: Mutagenesis, In Vivo, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Construct