Structured Review

Santa Cruz Biotechnology anti rarα
Increase in <t>RARα</t> at uterine implantation sites following pCR3.1-cyp26a1 immunization during mice peri-implantation and further certainty, the influence of RARα on Th17 cells at uterine implantation sites by tail vein injection of RARα specific agonist and antagonist during mice peri-implantation. (A) RARα levels were significantly increased at uterine implantation sites based on Western blotting. β-actin was used as a loading control. (B) RARα levels were obviously increased at implantation sites of D5 pregnancy based on immunohistochemistry following pCR3.1-cyp26a1 immunization; bars = 200 and 25 μm. The data were derived from three separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. E, embryo; S, uterine stroma; LE, Luminal epithelium; GE, Glandular epithelium. (C) RARα and RORγt protein expression changes in uterus on D5 of pregnancy in response to administration of agonist (Am-580) and antagonist (Ro-41-5253) of RARα. RARα levels were significantly increased by the activation of Am-580 and significantly reduced by the inhibition of Ro-41-5253 at uterine implantation sites based on western blotting of pregnancy D5. RORγt levels just took the opposite changes on RARα. β-actin was used as a loading control. The administration (125 μg/mouse) of RARα agonist (Am-580) or antagonist (Ro-41-5253) was performed on D3 of pregnancy through tail vein injection; ** P
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1) Product Images from "Regulation of cyp26a1 on Th17 cells in mouse peri-implantation"

Article Title: Regulation of cyp26a1 on Th17 cells in mouse peri-implantation

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12196

Increase in RARα at uterine implantation sites following pCR3.1-cyp26a1 immunization during mice peri-implantation and further certainty, the influence of RARα on Th17 cells at uterine implantation sites by tail vein injection of RARα specific agonist and antagonist during mice peri-implantation. (A) RARα levels were significantly increased at uterine implantation sites based on Western blotting. β-actin was used as a loading control. (B) RARα levels were obviously increased at implantation sites of D5 pregnancy based on immunohistochemistry following pCR3.1-cyp26a1 immunization; bars = 200 and 25 μm. The data were derived from three separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. E, embryo; S, uterine stroma; LE, Luminal epithelium; GE, Glandular epithelium. (C) RARα and RORγt protein expression changes in uterus on D5 of pregnancy in response to administration of agonist (Am-580) and antagonist (Ro-41-5253) of RARα. RARα levels were significantly increased by the activation of Am-580 and significantly reduced by the inhibition of Ro-41-5253 at uterine implantation sites based on western blotting of pregnancy D5. RORγt levels just took the opposite changes on RARα. β-actin was used as a loading control. The administration (125 μg/mouse) of RARα agonist (Am-580) or antagonist (Ro-41-5253) was performed on D3 of pregnancy through tail vein injection; ** P
Figure Legend Snippet: Increase in RARα at uterine implantation sites following pCR3.1-cyp26a1 immunization during mice peri-implantation and further certainty, the influence of RARα on Th17 cells at uterine implantation sites by tail vein injection of RARα specific agonist and antagonist during mice peri-implantation. (A) RARα levels were significantly increased at uterine implantation sites based on Western blotting. β-actin was used as a loading control. (B) RARα levels were obviously increased at implantation sites of D5 pregnancy based on immunohistochemistry following pCR3.1-cyp26a1 immunization; bars = 200 and 25 μm. The data were derived from three separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. E, embryo; S, uterine stroma; LE, Luminal epithelium; GE, Glandular epithelium. (C) RARα and RORγt protein expression changes in uterus on D5 of pregnancy in response to administration of agonist (Am-580) and antagonist (Ro-41-5253) of RARα. RARα levels were significantly increased by the activation of Am-580 and significantly reduced by the inhibition of Ro-41-5253 at uterine implantation sites based on western blotting of pregnancy D5. RORγt levels just took the opposite changes on RARα. β-actin was used as a loading control. The administration (125 μg/mouse) of RARα agonist (Am-580) or antagonist (Ro-41-5253) was performed on D3 of pregnancy through tail vein injection; ** P

Techniques Used: Mouse Assay, Injection, Western Blot, Immunohistochemistry, Derivative Assay, Expressing, Activation Assay, Inhibition

2) Product Images from "Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription"

Article Title: Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq023

Nuclear receptors and RNA Pol-II interact with NIS intronic elements in a dynamic manner during the initiation of transcription. MCF-7 cells grown in steroid-free and phenol-red-free DMEM were treated either with DMSO (time 0) or with 1 µM tRA for 15, 30 and 60 min and used for ChIP analysis using RARα and RXR and RNA Pol-II specific antibodies. ( A ) DNA isolated from immunocomplexes was used for quantitative PCR using primers described in Supplementary Table S3 . Ct values were normalized to background levels of bead-only controls. Data are represented as fold enrichment compared to IgG control. ( B ) mRNA was isolated from a fraction of the cells used for ChIP analysis above. Two micrograms of total RNA was converted to cDNA and used for quantitative real-time PCR. Expression was normalized to the levels of GAPDH using the ΔΔCt method and presented as relative fold induction compared to DMSO-treated samples. ( C ) Schematic representation of ChIP data depicting the events of transcription initiation of NIS in response to tRA stimulation. Arrow heads indicate the position of the intronic element investigated, vertical small lines represent NIS exons, numbers below indicate introns studied, asterisks above numbers indicate identical sequences. Parallelograms with question mark represent unidentified interacting proteins. DR, direct repeat; ER, everted repeat. Statistical significance was determined by performing the Student’s t -test ( Figure 7 A) or paired Student’s t -test ( Figure 7 C) using a 95% confidence interval; P -values
Figure Legend Snippet: Nuclear receptors and RNA Pol-II interact with NIS intronic elements in a dynamic manner during the initiation of transcription. MCF-7 cells grown in steroid-free and phenol-red-free DMEM were treated either with DMSO (time 0) or with 1 µM tRA for 15, 30 and 60 min and used for ChIP analysis using RARα and RXR and RNA Pol-II specific antibodies. ( A ) DNA isolated from immunocomplexes was used for quantitative PCR using primers described in Supplementary Table S3 . Ct values were normalized to background levels of bead-only controls. Data are represented as fold enrichment compared to IgG control. ( B ) mRNA was isolated from a fraction of the cells used for ChIP analysis above. Two micrograms of total RNA was converted to cDNA and used for quantitative real-time PCR. Expression was normalized to the levels of GAPDH using the ΔΔCt method and presented as relative fold induction compared to DMSO-treated samples. ( C ) Schematic representation of ChIP data depicting the events of transcription initiation of NIS in response to tRA stimulation. Arrow heads indicate the position of the intronic element investigated, vertical small lines represent NIS exons, numbers below indicate introns studied, asterisks above numbers indicate identical sequences. Parallelograms with question mark represent unidentified interacting proteins. DR, direct repeat; ER, everted repeat. Statistical significance was determined by performing the Student’s t -test ( Figure 7 A) or paired Student’s t -test ( Figure 7 C) using a 95% confidence interval; P -values

Techniques Used: Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction, Expressing

RARα and RXR interacts with the novel intronic RARE in gel retardation assays. Nuclear extracts from E2-treated MCF-7 cells were incubated with biotin labeled oligonucleotide probes representing a consensus RARE, the wild-type DR2-2 or the mutant variant DR2-2mut (DR2-2-Mut1-S in Supplementary Table S2 ). Samples were resolved on a 6% non-denaturing polyacrylamide gel in TBE, transferred to Hybond N+ membranes and then incubated with streptavidin, and biotin-labeled DNA probes were detected by chemiluminescence. The name of the probe used in each binding reaction is indicated on the top of each panel. All binding reactions were competed with 200-fold molar excess of the corresponding unlabeled probe. Arrows point out the nuclear receptor–DNA complexes, while arrowheads point out the super shift. The asterisk indicates a DR2-2 independent interaction with nuclear proteins. Labeled probes were incubated in the absence of nuclear extract (lanes 1, 6 and 11), in the presence of nuclear extract alone (lanes 2, 7 and 12), nuclear extract together with an excess of competing unlabeled probe (lanes 3, 8 and 13), in the presence of RARα antibodies (lanes 4, 9 and 14), or in the presence of RXR antibodies (lanes 5, 10 and 15). Experiments were repeated at least three times and a representative result is shown.
Figure Legend Snippet: RARα and RXR interacts with the novel intronic RARE in gel retardation assays. Nuclear extracts from E2-treated MCF-7 cells were incubated with biotin labeled oligonucleotide probes representing a consensus RARE, the wild-type DR2-2 or the mutant variant DR2-2mut (DR2-2-Mut1-S in Supplementary Table S2 ). Samples were resolved on a 6% non-denaturing polyacrylamide gel in TBE, transferred to Hybond N+ membranes and then incubated with streptavidin, and biotin-labeled DNA probes were detected by chemiluminescence. The name of the probe used in each binding reaction is indicated on the top of each panel. All binding reactions were competed with 200-fold molar excess of the corresponding unlabeled probe. Arrows point out the nuclear receptor–DNA complexes, while arrowheads point out the super shift. The asterisk indicates a DR2-2 independent interaction with nuclear proteins. Labeled probes were incubated in the absence of nuclear extract (lanes 1, 6 and 11), in the presence of nuclear extract alone (lanes 2, 7 and 12), nuclear extract together with an excess of competing unlabeled probe (lanes 3, 8 and 13), in the presence of RARα antibodies (lanes 4, 9 and 14), or in the presence of RXR antibodies (lanes 5, 10 and 15). Experiments were repeated at least three times and a representative result is shown.

Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Labeling, Mutagenesis, Variant Assay, Binding Assay

RARα and RXR occupy the novel intronic element in vivo . MCF-7 cells grown in DMEM were treated with 1 µM tRA and used for ChIP analysis using RARα and RXR specific antibodies. ( A ) DNA isolated from immunocomplexes was used as a template for PCR amplification using primers specific for DR2-2. PCR products were resolved on a 2% agarose gel (containing Ethidium bromide) and visualized on a UV transilluminator, RARα; anti-RAR-α Ab precipitated DNA, RXR; anti-RXR Ab precipitated DNA, FGFR1; anti-FGFR1 Ab precipitated DNA, No Ab; bead-only control, and -PCR is a negative control with H 2 O as a template. ( B ) Quantitative PCR was performed using immunoprecipitated DNA using DR2-1 and DR2-2 specific primers. Ct values were normalized to background levels of bead-only controls (No Ab) using 2 (δCt) . Data are represented as fold enrichment compared to IgG control. * P -values were calculated using Student’s t -test (average of three experiments) with 95% confidence interval, values
Figure Legend Snippet: RARα and RXR occupy the novel intronic element in vivo . MCF-7 cells grown in DMEM were treated with 1 µM tRA and used for ChIP analysis using RARα and RXR specific antibodies. ( A ) DNA isolated from immunocomplexes was used as a template for PCR amplification using primers specific for DR2-2. PCR products were resolved on a 2% agarose gel (containing Ethidium bromide) and visualized on a UV transilluminator, RARα; anti-RAR-α Ab precipitated DNA, RXR; anti-RXR Ab precipitated DNA, FGFR1; anti-FGFR1 Ab precipitated DNA, No Ab; bead-only control, and -PCR is a negative control with H 2 O as a template. ( B ) Quantitative PCR was performed using immunoprecipitated DNA using DR2-1 and DR2-2 specific primers. Ct values were normalized to background levels of bead-only controls (No Ab) using 2 (δCt) . Data are represented as fold enrichment compared to IgG control. * P -values were calculated using Student’s t -test (average of three experiments) with 95% confidence interval, values

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Isolation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Negative Control, Real-time Polymerase Chain Reaction, Immunoprecipitation

3) Product Images from "Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *"

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.538777

PLZF-RARα promotes cell proliferation but does not induce apoptosis. A, FACS analysis of HEK293 and HCT116 cells transfected with either a pcDNA3 or pSG5-PLZF-RARα plasmid. B , MTT assay of cell proliferation. HEK293 and HCT116 cells transfected
Figure Legend Snippet: PLZF-RARα promotes cell proliferation but does not induce apoptosis. A, FACS analysis of HEK293 and HCT116 cells transfected with either a pcDNA3 or pSG5-PLZF-RARα plasmid. B , MTT assay of cell proliferation. HEK293 and HCT116 cells transfected

Techniques Used: FACS, Transfection, Plasmid Preparation, MTT Assay

Transcription repression of CDKN1A by PLZF-RARα can be independent of p53. A , transcription assays. HCT116 p53 +/+ and p53 −/− cells were transiently co-transfected with a PLZF-RARα expression vector and a pGL2-CDKN1A-Luc
Figure Legend Snippet: Transcription repression of CDKN1A by PLZF-RARα can be independent of p53. A , transcription assays. HCT116 p53 +/+ and p53 −/− cells were transiently co-transfected with a PLZF-RARα expression vector and a pGL2-CDKN1A-Luc

Techniques Used: Transfection, Expressing, Plasmid Preparation

Hypothetical model for transcriptional regulation of CDKN1A and TP53 by PLZF-RARα and post-translational ubiquitination of p53. A , PLZF-RARα represses all four genes in the p53 pathway. ⊢, transcriptional repression; ↔,
Figure Legend Snippet: Hypothetical model for transcriptional regulation of CDKN1A and TP53 by PLZF-RARα and post-translational ubiquitination of p53. A , PLZF-RARα represses all four genes in the p53 pathway. ⊢, transcriptional repression; ↔,

Techniques Used:

PLZF-RARα stimulates cell proliferation and represses CDKN1A transcription in HL-60 cells through inhibitory histone modifications and DNA methylation. A , MTT assay of cell proliferation. HL-60 cells transfected with either pcDNA3 or pSG5-PLZF-RARα
Figure Legend Snippet: PLZF-RARα stimulates cell proliferation and represses CDKN1A transcription in HL-60 cells through inhibitory histone modifications and DNA methylation. A , MTT assay of cell proliferation. HL-60 cells transfected with either pcDNA3 or pSG5-PLZF-RARα

Techniques Used: DNA Methylation Assay, MTT Assay, Transfection

PLZF-RARα represses CDKN1A gene transcription through binding competition with p53, TP53 transcriptional repression, and increased p53 ubiquitination. A , structure of the human CDKN1A gene promoter. The arrows at the p53 binding elements indicate
Figure Legend Snippet: PLZF-RARα represses CDKN1A gene transcription through binding competition with p53, TP53 transcriptional repression, and increased p53 ubiquitination. A , structure of the human CDKN1A gene promoter. The arrows at the p53 binding elements indicate

Techniques Used: Binding Assay

PLZF-RARα represses the transcription of p53 pathway genes in HEK293 cells. A, transient transcription assays for the ARF, MDM2, TP53 , and CDKN1A genes of the p53 pathway. The PLZF-RARα expression vector and promoter-luciferase fusion
Figure Legend Snippet: PLZF-RARα represses the transcription of p53 pathway genes in HEK293 cells. A, transient transcription assays for the ARF, MDM2, TP53 , and CDKN1A genes of the p53 pathway. The PLZF-RARα expression vector and promoter-luciferase fusion

Techniques Used: Expressing, Plasmid Preparation, Luciferase

PLZF-RARα represses the transcription of CDKN1A by binding to its RARE in vitro and in vivo . A , transcription assay for the CDKN1A promoter in the presence of PLZF-RARα and ATRA or EtOH control in HEK293 cells. Cells were transiently co-transfected
Figure Legend Snippet: PLZF-RARα represses the transcription of CDKN1A by binding to its RARE in vitro and in vivo . A , transcription assay for the CDKN1A promoter in the presence of PLZF-RARα and ATRA or EtOH control in HEK293 cells. Cells were transiently co-transfected

Techniques Used: Binding Assay, In Vitro, In Vivo, Transfection

Proliferation of HL-60 cells is increased by ectopic PLZF-RARα and decreased by TSA, 5-aza-2′-deoxycytidine, ATRA, or any combination thereof. Transcriptional repression of CDKN1A by PLZF-RARα is derepressed by the reagents. A–D
Figure Legend Snippet: Proliferation of HL-60 cells is increased by ectopic PLZF-RARα and decreased by TSA, 5-aza-2′-deoxycytidine, ATRA, or any combination thereof. Transcriptional repression of CDKN1A by PLZF-RARα is derepressed by the reagents. A–D

Techniques Used:

PLZF-RARα represses transcription of CDKN1A epigenetically by histone deacetylation and DNA methylation. A , structure of the human CDKN1A gene promoter. The arrows indicate the locations of the qChIP-PCR primer binding sites. B , qChIP assays showing
Figure Legend Snippet: PLZF-RARα represses transcription of CDKN1A epigenetically by histone deacetylation and DNA methylation. A , structure of the human CDKN1A gene promoter. The arrows indicate the locations of the qChIP-PCR primer binding sites. B , qChIP assays showing

Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Binding Assay

PLZF-RARα represses CDKN1A transcription by competing with Sp1 for binding to proximal promoter GC-boxes 3, 4, and 5/6, in vitro and in vivo . A , structure of the human CDKN1A promoter. The arrows indicate the binding positions of the qChIP oligonucleotide
Figure Legend Snippet: PLZF-RARα represses CDKN1A transcription by competing with Sp1 for binding to proximal promoter GC-boxes 3, 4, and 5/6, in vitro and in vivo . A , structure of the human CDKN1A promoter. The arrows indicate the binding positions of the qChIP oligonucleotide

Techniques Used: Binding Assay, In Vitro, In Vivo

PLZF-RARα represses transcriptional activation of CDKN1A by p53. A , transient transcription assays. HCT116 cells were transiently co-transfected with a PLZF-RARα expression vector and a pGL2-CDKN1A-Luc (−2.3 kb) reporter plasmid,
Figure Legend Snippet: PLZF-RARα represses transcriptional activation of CDKN1A by p53. A , transient transcription assays. HCT116 cells were transiently co-transfected with a PLZF-RARα expression vector and a pGL2-CDKN1A-Luc (−2.3 kb) reporter plasmid,

Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation

4) Product Images from "The Ski protein can inhibit ligand induced RAR? and HDAC3 degradation in the Retinoic acid signaling pathway"

Article Title: The Ski protein can inhibit ligand induced RAR? and HDAC3 degradation in the Retinoic acid signaling pathway

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2009.03.141

RA-induced RARα degradation is important for optimal RARα-mediated transactivation
Figure Legend Snippet: RA-induced RARα degradation is important for optimal RARα-mediated transactivation

Techniques Used:

Ski expression inhibits RA-induced RARα degradation
Figure Legend Snippet: Ski expression inhibits RA-induced RARα degradation

Techniques Used: Expressing

Ski and RARα are in the same complex in both the presence and absence of RA
Figure Legend Snippet: Ski and RARα are in the same complex in both the presence and absence of RA

Techniques Used:

5) Product Images from "Targeting the Acute Promyelocytic Leukemia-Associated Fusion Proteins PML/RAR? and PLZF/RAR? with Interfering Peptides"

Article Title: Targeting the Acute Promyelocytic Leukemia-Associated Fusion Proteins PML/RAR? and PLZF/RAR? with Interfering Peptides

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048636

PCC and POZ reverse the proliferation capacity of PML/RARα- and PLZF/RARα-positive murine hematopoietic stem cells. A , Schematic diagram of the experimental conditions. Sca1 + /lin − bone marrow cells were isolated and infected with retroviral vectors containing PML/RARα or PLZF/RARα and GFP or PCC/POZ-GFP. Infected cells were plated in methylcellulose on day 3. Colony counts and replating were measured every 10 days. B and C , Colony count of Sca1 + /lin − bone marrow cells infected with PML/RARα ( B ) or PLZF/RARα ( C ) and GFP or PCC/POZ-GFP cultured in methylcellulose. Mock: empty vector.
Figure Legend Snippet: PCC and POZ reverse the proliferation capacity of PML/RARα- and PLZF/RARα-positive murine hematopoietic stem cells. A , Schematic diagram of the experimental conditions. Sca1 + /lin − bone marrow cells were isolated and infected with retroviral vectors containing PML/RARα or PLZF/RARα and GFP or PCC/POZ-GFP. Infected cells were plated in methylcellulose on day 3. Colony counts and replating were measured every 10 days. B and C , Colony count of Sca1 + /lin − bone marrow cells infected with PML/RARα ( B ) or PLZF/RARα ( C ) and GFP or PCC/POZ-GFP cultured in methylcellulose. Mock: empty vector.

Techniques Used: Periodic Counter-current Chromatography, Isolation, Infection, Cell Culture, Plasmid Preparation

PCC and POZ influences on the HMW complex formation of PML/RARα and PLZF/RARα, respectively. A and B , Size-exclusion chromatography fractions of in vitro translated PML/RARα ( A ) or PLZF/RARα ( B ) and HA-tagged peptide. Fractions were analyzed by western blot (on the right) and probed against RARα. The densitometric analysis of the western blot is shown on the left side as a percentage of the entire signal. C and D , Size-exclusion chromatography of the Phoenix whole cell lysates overexpressing PML/RARα ( C ) or PLZF/RARα ( D ) and GFP-tagged peptide. Fractions were analyzed by western blot (WB) and probed against RARα (α-RARα) (lower part). The densitometric analysis of the western blot is shown in the upper part as a percentage of the entire signal. HA-mock: control empty vector. Input (I), eluted fractions 14 to 29. The numbers above the arrows represent the molecular weight (KD) of the proteins used for the MW calibration: 136 KD for the BSA dimer, 440 KD for Ferritin, and 670 KD for Thyroglobulin.
Figure Legend Snippet: PCC and POZ influences on the HMW complex formation of PML/RARα and PLZF/RARα, respectively. A and B , Size-exclusion chromatography fractions of in vitro translated PML/RARα ( A ) or PLZF/RARα ( B ) and HA-tagged peptide. Fractions were analyzed by western blot (on the right) and probed against RARα. The densitometric analysis of the western blot is shown on the left side as a percentage of the entire signal. C and D , Size-exclusion chromatography of the Phoenix whole cell lysates overexpressing PML/RARα ( C ) or PLZF/RARα ( D ) and GFP-tagged peptide. Fractions were analyzed by western blot (WB) and probed against RARα (α-RARα) (lower part). The densitometric analysis of the western blot is shown in the upper part as a percentage of the entire signal. HA-mock: control empty vector. Input (I), eluted fractions 14 to 29. The numbers above the arrows represent the molecular weight (KD) of the proteins used for the MW calibration: 136 KD for the BSA dimer, 440 KD for Ferritin, and 670 KD for Thyroglobulin.

Techniques Used: Periodic Counter-current Chromatography, Size-exclusion Chromatography, In Vitro, Western Blot, Plasmid Preparation, Molecular Weight

PCC and POZ bind to PML/RARα and PLZF/RARα, respectively. A , Theory for the peptide binding to X-RARα, as exemplified by POZ and PLZF/RARα. By binding the oligomerization domain of PLZF/RARα, POZ disables the self-oligomerization and the high-molecular-weight complex formation. B , C , HA-tagged peptides were co-expressed with PML/RARα or PLZF/RARα in the 293 cells through the transfection of the corresponding combination of pCDNA3 vectors with calcium phosphate. An HA-empty plasmid and an HA-tagged BCC (coiled-coil domain of BCR) were used as a specificity control. In B , PCC was transfected at two different concentrations, 5 and 10 µG DNA (first and second BCC lane, respectively). Immunoprecipitation (IP) was performed with an anti-HA matrix. Western blots were probed with α-HA and α-RARα antibodies.
Figure Legend Snippet: PCC and POZ bind to PML/RARα and PLZF/RARα, respectively. A , Theory for the peptide binding to X-RARα, as exemplified by POZ and PLZF/RARα. By binding the oligomerization domain of PLZF/RARα, POZ disables the self-oligomerization and the high-molecular-weight complex formation. B , C , HA-tagged peptides were co-expressed with PML/RARα or PLZF/RARα in the 293 cells through the transfection of the corresponding combination of pCDNA3 vectors with calcium phosphate. An HA-empty plasmid and an HA-tagged BCC (coiled-coil domain of BCR) were used as a specificity control. In B , PCC was transfected at two different concentrations, 5 and 10 µG DNA (first and second BCC lane, respectively). Immunoprecipitation (IP) was performed with an anti-HA matrix. Western blots were probed with α-HA and α-RARα antibodies.

Techniques Used: Periodic Counter-current Chromatography, Binding Assay, Molecular Weight, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

PCC and POZ reverse the differentiation block of PML/RARα- and PLZF/RARα-positive murine hematopoietic stem cells. A , Schematic diagram of the experiment conditions. Sca1 + /lin - murine bone marrow cells were isolated and infected with retroviral vectors containing PML/RARα or PLZF/RARα and GFP or PCC/POZ-GFP and then analyzed for differentiation. B , The infection efficiency, measured as the percentage of the GFP-positive cells to the retrovirally infected cells. C , Reverse transcriptase-PCR of the retrovirus-infected Sca + /lin − bone marrow cells for PML/RARα and PLZF/RARα. Control: β-Actin. D , GIEMSA staining of Sca + /lin − bone marrow cells seven days after infection with retroviral vectors. E , FACS analysis of the Sca1 + /lin − bone marrow cells infected with PML/RARα and GFP or PCC/POZ-GFP. Mock: empty vector. F , FACS analysis of the Sca1 + /lin − bone marrow cells infected with PLZF/RARα and GFP or PCC/POZ-GFP. Mock: empty vector.
Figure Legend Snippet: PCC and POZ reverse the differentiation block of PML/RARα- and PLZF/RARα-positive murine hematopoietic stem cells. A , Schematic diagram of the experiment conditions. Sca1 + /lin - murine bone marrow cells were isolated and infected with retroviral vectors containing PML/RARα or PLZF/RARα and GFP or PCC/POZ-GFP and then analyzed for differentiation. B , The infection efficiency, measured as the percentage of the GFP-positive cells to the retrovirally infected cells. C , Reverse transcriptase-PCR of the retrovirus-infected Sca + /lin − bone marrow cells for PML/RARα and PLZF/RARα. Control: β-Actin. D , GIEMSA staining of Sca + /lin − bone marrow cells seven days after infection with retroviral vectors. E , FACS analysis of the Sca1 + /lin − bone marrow cells infected with PML/RARα and GFP or PCC/POZ-GFP. Mock: empty vector. F , FACS analysis of the Sca1 + /lin − bone marrow cells infected with PLZF/RARα and GFP or PCC/POZ-GFP. Mock: empty vector.

Techniques Used: Periodic Counter-current Chromatography, Blocking Assay, Isolation, Infection, Polymerase Chain Reaction, Staining, FACS, Plasmid Preparation

PM/RARα and PLZF/RARα, degrade in the presence of PCC and POZ, respectively. A and B , Western blot of the whole cell lysates of PML/RARα- ( A ) or PLZF/RARα ( B )-positive Phoenix and BA/F3 cells probed against RARα (α-RARα) and GFP (α-GFP). C and D , BA/F3 cells were treated with 10 µM MG132 or 20 µM Calpain I for 16 h. Control: empty vector; tubulin: loading control. E . BA/F3 cells were treated with 500 nM Lactacystin for 24 h. Control: empty vector; GAPDH: loading control. F , The infection efficiency, measured as the percentage of GFP-positive cells, of the Ba/F3 cells after infection with PINCO (control) or PIDE carrying PLZF/RARα alone or in combination with GFP, GFP-POZ or GFP-PCC or PML/RARα alone or in combination with GFP, GFP-POZ or GFP-PCC peptides, as indicated. G , Western blot of the Phoenix whole cell lysate expressing PML/RARα or a sumoylation-deficient mutant (PML3-160/RARα) PCC-GFP or GFP, probed with α-GFP, α-RARα and α-GPADH as a loading control. The image shown is a representative of three separate experiments.
Figure Legend Snippet: PM/RARα and PLZF/RARα, degrade in the presence of PCC and POZ, respectively. A and B , Western blot of the whole cell lysates of PML/RARα- ( A ) or PLZF/RARα ( B )-positive Phoenix and BA/F3 cells probed against RARα (α-RARα) and GFP (α-GFP). C and D , BA/F3 cells were treated with 10 µM MG132 or 20 µM Calpain I for 16 h. Control: empty vector; tubulin: loading control. E . BA/F3 cells were treated with 500 nM Lactacystin for 24 h. Control: empty vector; GAPDH: loading control. F , The infection efficiency, measured as the percentage of GFP-positive cells, of the Ba/F3 cells after infection with PINCO (control) or PIDE carrying PLZF/RARα alone or in combination with GFP, GFP-POZ or GFP-PCC or PML/RARα alone or in combination with GFP, GFP-POZ or GFP-PCC peptides, as indicated. G , Western blot of the Phoenix whole cell lysate expressing PML/RARα or a sumoylation-deficient mutant (PML3-160/RARα) PCC-GFP or GFP, probed with α-GFP, α-RARα and α-GPADH as a loading control. The image shown is a representative of three separate experiments.

Techniques Used: Periodic Counter-current Chromatography, Western Blot, Plasmid Preparation, Infection, Expressing, Mutagenesis

6) Product Images from "Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system"

Article Title: Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system

Journal: Mucosal immunology

doi: 10.1038/mi.2017.91

Dendritic cell numbers are decreased in RARα villin mice. Cell suspensions from small intestinal lamina propria were analyzed by FACS. ( a ) Analysis of single live CD45 + events from the flow cytometry data of SI lamina propria isolated form control or RARα villin mice using the t-distributed stochastic linear embedding (tSNE) algorithm (one representative analysis of two) ( b ) Representative dot plots showing total dendritic cells (left column) and subsets defined by CD103 and CD11b expression (middle column) as well as macrophages defined by CD64 (right column). ( c ) Quantification of the frequencies of dendritic cells (DCs) and the CD103 + subset. ( n = 4; 2 experiments) ( d ) Expression of CD11c in the small intestine was determined by immunohistochemistry. Representative images of 6 images/mouse (n = 3 mice). ( e ) Representative dot plot and quantification of RA-producing dendritic cells in the small intestine lamina propria determined by ALDEFLUOR (ALDE) ( n = 4; 2 experiments). * P
Figure Legend Snippet: Dendritic cell numbers are decreased in RARα villin mice. Cell suspensions from small intestinal lamina propria were analyzed by FACS. ( a ) Analysis of single live CD45 + events from the flow cytometry data of SI lamina propria isolated form control or RARα villin mice using the t-distributed stochastic linear embedding (tSNE) algorithm (one representative analysis of two) ( b ) Representative dot plots showing total dendritic cells (left column) and subsets defined by CD103 and CD11b expression (middle column) as well as macrophages defined by CD64 (right column). ( c ) Quantification of the frequencies of dendritic cells (DCs) and the CD103 + subset. ( n = 4; 2 experiments) ( d ) Expression of CD11c in the small intestine was determined by immunohistochemistry. Representative images of 6 images/mouse (n = 3 mice). ( e ) Representative dot plot and quantification of RA-producing dendritic cells in the small intestine lamina propria determined by ALDEFLUOR (ALDE) ( n = 4; 2 experiments). * P

Techniques Used: Mouse Assay, FACS, Flow Cytometry, Cytometry, Isolation, Expressing, Immunohistochemistry

RARα signaling on IECs regulates intestinal secretory cell differentiation and immunological fitness. Scheme showing the proposed model in which RARα expression in proliferating cells (transit amplifying progenitors) restrict secretory cell differentiation and Reg3g expression which is associated to proper microbiota composition and immune cell development.
Figure Legend Snippet: RARα signaling on IECs regulates intestinal secretory cell differentiation and immunological fitness. Scheme showing the proposed model in which RARα expression in proliferating cells (transit amplifying progenitors) restrict secretory cell differentiation and Reg3g expression which is associated to proper microbiota composition and immune cell development.

Techniques Used: Cell Differentiation, Expressing

RARα deficiency in the epithelial compartment results in defective clearance of C. rodentium. ( a ) CFU from control and RARα villin mice infected with C. rodentium . ( n = 4; 2 experiments) ( b ). Bioluminescence of cultured stool samples (colon), spleen, and MLN cell suspensions. ( c ) Bioluminescence of whole animal. ( d ) Body weight loss curves from control and RARα villin mice infected with C. rodentium (2 x10 9 CFU). ( n = 5; 2 experiments *** P
Figure Legend Snippet: RARα deficiency in the epithelial compartment results in defective clearance of C. rodentium. ( a ) CFU from control and RARα villin mice infected with C. rodentium . ( n = 4; 2 experiments) ( b ). Bioluminescence of cultured stool samples (colon), spleen, and MLN cell suspensions. ( c ) Bioluminescence of whole animal. ( d ) Body weight loss curves from control and RARα villin mice infected with C. rodentium (2 x10 9 CFU). ( n = 5; 2 experiments *** P

Techniques Used: Mouse Assay, Infection, Cell Culture

RARα controls epithelial homeostasis. ( a–b ) Mucins-containing goblet cells were stained with Alcian Blue and their number per villus determined. ( c–d ) Paneth cells were immunostained with anti-lysozyme and their number per villus and position along the crypt-villus axis determined ( n = 7–19 villus/mouse). ( e–f ) Enteroendocrine cells were immunostained with anti-Chromogranin A and their number per villus determined ( n = 7–12 villus/mouse). Data in ( a–b ) are representative of three mice/genotype. ** P
Figure Legend Snippet: RARα controls epithelial homeostasis. ( a–b ) Mucins-containing goblet cells were stained with Alcian Blue and their number per villus determined. ( c–d ) Paneth cells were immunostained with anti-lysozyme and their number per villus and position along the crypt-villus axis determined ( n = 7–19 villus/mouse). ( e–f ) Enteroendocrine cells were immunostained with anti-Chromogranin A and their number per villus determined ( n = 7–12 villus/mouse). Data in ( a–b ) are representative of three mice/genotype. ** P

Techniques Used: Staining, Mouse Assay

RARα modulates differentiation within the secretory branch through KLF4. ( a ) Proliferative cells were identified by BrdU incorporation in parallel with lysozyme or muc-2 to detect paneth cells (left) and goblet cells (right), respectively. ( b–c ) KLF4 expression was measured in the distal small intestine ( b ) and colon ( c ) by immunohistochemistry and number of positive nuclei were counted per intestinal gland. 7–10 crypts were counted per intestine section in 2 mice per genotype. ( d ) RT-qPCR analysis of RAR target gene cyp26a1 and the transcription factor klf4 in zebrafish embryos treated with either vehicle or 1μM RA from 72 hours post-fertilization (hpf) till 108 hpf. Each dot represents a pool of 20 embryos. The mRNA expression was normalized to that of ef1α . ( e ) Whole-mount alcian blue staining of zebrafish embryos treated with either vehicle or 1μM RA from 72 hpf till 108 hpf (images). The graph represent quantification of alcian blue positive cells per intestine ( n =8 per group). * P
Figure Legend Snippet: RARα modulates differentiation within the secretory branch through KLF4. ( a ) Proliferative cells were identified by BrdU incorporation in parallel with lysozyme or muc-2 to detect paneth cells (left) and goblet cells (right), respectively. ( b–c ) KLF4 expression was measured in the distal small intestine ( b ) and colon ( c ) by immunohistochemistry and number of positive nuclei were counted per intestinal gland. 7–10 crypts were counted per intestine section in 2 mice per genotype. ( d ) RT-qPCR analysis of RAR target gene cyp26a1 and the transcription factor klf4 in zebrafish embryos treated with either vehicle or 1μM RA from 72 hours post-fertilization (hpf) till 108 hpf. Each dot represents a pool of 20 embryos. The mRNA expression was normalized to that of ef1α . ( e ) Whole-mount alcian blue staining of zebrafish embryos treated with either vehicle or 1μM RA from 72 hpf till 108 hpf (images). The graph represent quantification of alcian blue positive cells per intestine ( n =8 per group). * P

Techniques Used: BrdU Incorporation Assay, Expressing, Immunohistochemistry, Mouse Assay, Quantitative RT-PCR, Staining

Dysbiosis in RARα villin mice. ( a ) qPCR analysis from FACS-sorted epithelial cells (CD45 neg EpCAM + ) obtained from the proximal or distal small intestine of either control or RARα villin mice. Data shows transcript levels as arbitrary units (A.U.) respect to hprt ( n = 3 mice). ( b ) Fluorescence in situ hybridization of universal 16S ribosomal RNA in DAPI-stained ileal tissues from control and RARα villin mice. Original magnification, 10X. One representative image of 3–6 images/mouse ( n = 2 mice). ( c ) 16S rDNA mean intensities (sum of pixel intensities/number of pixels) are reported. Scale bar, 10 μm. ( d ) qPCR analysis shows arbitrary units (A.U.) of lactobacillus, enterobacterae (entero), bacteroides, clostridia and segmented filamentous bacteria (SFB) relative to universal 16S levels. Bacterial DNA was isolated from luminal stool obtained from the small intestine (SI) or colon ( n = 3; 2 experiments). * P
Figure Legend Snippet: Dysbiosis in RARα villin mice. ( a ) qPCR analysis from FACS-sorted epithelial cells (CD45 neg EpCAM + ) obtained from the proximal or distal small intestine of either control or RARα villin mice. Data shows transcript levels as arbitrary units (A.U.) respect to hprt ( n = 3 mice). ( b ) Fluorescence in situ hybridization of universal 16S ribosomal RNA in DAPI-stained ileal tissues from control and RARα villin mice. Original magnification, 10X. One representative image of 3–6 images/mouse ( n = 2 mice). ( c ) 16S rDNA mean intensities (sum of pixel intensities/number of pixels) are reported. Scale bar, 10 μm. ( d ) qPCR analysis shows arbitrary units (A.U.) of lactobacillus, enterobacterae (entero), bacteroides, clostridia and segmented filamentous bacteria (SFB) relative to universal 16S levels. Bacterial DNA was isolated from luminal stool obtained from the small intestine (SI) or colon ( n = 3; 2 experiments). * P

Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, FACS, Fluorescence, In Situ Hybridization, Staining, Isolation

RARα deficiency results in altered intestinal immune development. ( a ) Expression of CD3 in the small intestine was determined by immunohistochemistry. Representative image of 6 images/mouse (n = 3 mice). ( b–c ) Representative dot plots showing total CD90 + cells ( b ) and T cells and ILCs within the CD90 + compartment (c) ( n = 3–4; 2 experiments). ( d ) Colon Swiss rolls showing immunohistochemistry for B220 in control and RARα villin mice. Data are representative of three mice/genotype. ( e ) Colon cell suspension staining for B220 and CD11c reveals decreased B cells in RARα villin mice ( n = 3; 2 experiments). * P
Figure Legend Snippet: RARα deficiency results in altered intestinal immune development. ( a ) Expression of CD3 in the small intestine was determined by immunohistochemistry. Representative image of 6 images/mouse (n = 3 mice). ( b–c ) Representative dot plots showing total CD90 + cells ( b ) and T cells and ILCs within the CD90 + compartment (c) ( n = 3–4; 2 experiments). ( d ) Colon Swiss rolls showing immunohistochemistry for B220 in control and RARα villin mice. Data are representative of three mice/genotype. ( e ) Colon cell suspension staining for B220 and CD11c reveals decreased B cells in RARα villin mice ( n = 3; 2 experiments). * P

Techniques Used: Expressing, Immunohistochemistry, Mouse Assay, Staining

RARα expression in intestinal epithelial cells from the small intestine and colon. ( a–b ) Frozen sections from the proximal, medial and distal small intestine ( a ) and proximal and distal colon ( b ) were stainied for RARα. Onsets show a digital magnification of the crypt within the respective boxes. ( c ) Cartoon showing the RARα expression pattern through the crypt-villi axis (Small intestine) or crypt (colon). One representative figure out of three experiments. ( d ) H E staining of distal small intestine sections of control and RARα villin mice. One representative figure out of three experiments. TA: transit amplifying. Scale bars 100uM
Figure Legend Snippet: RARα expression in intestinal epithelial cells from the small intestine and colon. ( a–b ) Frozen sections from the proximal, medial and distal small intestine ( a ) and proximal and distal colon ( b ) were stainied for RARα. Onsets show a digital magnification of the crypt within the respective boxes. ( c ) Cartoon showing the RARα expression pattern through the crypt-villi axis (Small intestine) or crypt (colon). One representative figure out of three experiments. ( d ) H E staining of distal small intestine sections of control and RARα villin mice. One representative figure out of three experiments. TA: transit amplifying. Scale bars 100uM

Techniques Used: Expressing, Staining, Mouse Assay

7) Product Images from "Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells"

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0096512

RA enhances GM-CSF-induced Aldh1a2 expression via the RARα/RXRα heterodimer bound to the RARE half-site. ( A ) Localization of the putative RARE half-sites ( underlined ) in Probe C and their mutants, Probe C(RARE-h mt1) and Probe C(RARE-h mt2), are shown. COS-7 cells were transfected with the 0.5 µg of pSG5-RARα and/or pSG5-RXRα. One day after transfection, cell lysates were subjected to DNAP assay using the biotinylated DNA probes. The precipitates were analyzed by Western blotting using anti-RARα( upper panel ) and RXRα ( lower panel ) Abs. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 100 nM RA. LE540 (1 µM) was added to the indicated cultures. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. ( C ) BM-DCs were cultured with ( closed circle ) or without ( open circle ) 10 ng/ml GM-CSF for 16 h in the presence of graded concentrations of RA. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. Asterisks indicate a significant difference (*** p
Figure Legend Snippet: RA enhances GM-CSF-induced Aldh1a2 expression via the RARα/RXRα heterodimer bound to the RARE half-site. ( A ) Localization of the putative RARE half-sites ( underlined ) in Probe C and their mutants, Probe C(RARE-h mt1) and Probe C(RARE-h mt2), are shown. COS-7 cells were transfected with the 0.5 µg of pSG5-RARα and/or pSG5-RXRα. One day after transfection, cell lysates were subjected to DNAP assay using the biotinylated DNA probes. The precipitates were analyzed by Western blotting using anti-RARα( upper panel ) and RXRα ( lower panel ) Abs. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 100 nM RA. LE540 (1 µM) was added to the indicated cultures. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. ( C ) BM-DCs were cultured with ( closed circle ) or without ( open circle ) 10 ng/ml GM-CSF for 16 h in the presence of graded concentrations of RA. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. Asterisks indicate a significant difference (*** p

Techniques Used: Expressing, Transfection, Western Blot, Generated, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity. ( A ) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3 . ( B ) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. ( C ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (* p
Figure Legend Snippet: Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity. ( A ) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3 . ( B ) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. ( C ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (* p

Techniques Used: Binding Assay, Activity Assay, Transfection, Sequencing, Plasmid Preparation, Expressing, Luciferase, Generated, Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

8) Product Images from "PU.1 controls the expression of long noncoding RNA HOTAIRM1 during granulocytic differentiation"

Article Title: PU.1 controls the expression of long noncoding RNA HOTAIRM1 during granulocytic differentiation

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-016-0274-1

Low HOTAIRM1 expression is attributed to the reduced PU.1 expression. a HOTAIRM1 and PU.1 mRNA expression levels in representative AML cell lines. b Low levels of HOTAIRM1 and PU.1 in APL patients, as compared with non-APL patients (GSE10358). c ChIP-qPCR showing the binding of PML-RARα on the HOTAIRM1 promoter, the PU.1 promoter (as a positive control), and the negative site. d The effect of PML-RARα on promoter activity of HOTAIRM1 . The pGL3-HOTAIRM1 plasmid was co-transfected with 200 ng expression vectors (pcDNA3.1 alone, PML-RARα alone, PU.1 alone, PU.1 and PML-RARα together, respectively) into HEK-293T cells. e The Pearson correlation between PU.1 and HOTAIRM1 expression in an AML patient cohort (GSE10358). f The up-regulation of HOTAIRM1 after PU.1 overexpression. NB4 cells were harvested at 48 h post-transduction with the MigR1 empty vector or the MigR1-PU.1 vector. The expression level of PU.1 was detected by western blotting. The mRNA expression levels of HOTAIRM1 and ITGAM were detected by RT-qPCR. GAPDH was used for normalization. The data represent the mean of three replicates ± SD. * p
Figure Legend Snippet: Low HOTAIRM1 expression is attributed to the reduced PU.1 expression. a HOTAIRM1 and PU.1 mRNA expression levels in representative AML cell lines. b Low levels of HOTAIRM1 and PU.1 in APL patients, as compared with non-APL patients (GSE10358). c ChIP-qPCR showing the binding of PML-RARα on the HOTAIRM1 promoter, the PU.1 promoter (as a positive control), and the negative site. d The effect of PML-RARα on promoter activity of HOTAIRM1 . The pGL3-HOTAIRM1 plasmid was co-transfected with 200 ng expression vectors (pcDNA3.1 alone, PML-RARα alone, PU.1 alone, PU.1 and PML-RARα together, respectively) into HEK-293T cells. e The Pearson correlation between PU.1 and HOTAIRM1 expression in an AML patient cohort (GSE10358). f The up-regulation of HOTAIRM1 after PU.1 overexpression. NB4 cells were harvested at 48 h post-transduction with the MigR1 empty vector or the MigR1-PU.1 vector. The expression level of PU.1 was detected by western blotting. The mRNA expression levels of HOTAIRM1 and ITGAM were detected by RT-qPCR. GAPDH was used for normalization. The data represent the mean of three replicates ± SD. * p

Techniques Used: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Positive Control, Activity Assay, Plasmid Preparation, Transfection, Over Expression, Transduction, Western Blot, Quantitative RT-PCR

9) Product Images from "Retinoic Acid Activates Monoamine Oxidase B Promoter in Human Neuronal Cells *"

Article Title: Retinoic Acid Activates Monoamine Oxidase B Promoter in Human Neuronal Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M901779200

RARα interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.
Figure Legend Snippet: RARα interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.

Techniques Used: Recombinant, Incubation, Immunoprecipitation, Western Blot, Negative Control, Co-Immunoprecipitation Assay, In Vitro, Binding Assay

10) Product Images from "BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid"

Article Title: BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121088

RARα fails to bind the 5′ regulatory regions of the mouse CCR9 and Itg-α4 genes in BATF deficiency. WT or BATF KO CD4 + T cells were activated for 4–5 d with concanavalin A, IL-2, and RA. (A) Expression of nuclear RAR genes in BATF KO CD4 + T cells was examined by quantitative RT-PCR. Normalized values to β-actin levels are shown. (B and C) The binding of RARα and BATF and histone H4 acetylation on the CCR9 gene (B) or the Itg-α4 gene (C) were assessed by ChIP assay. Representative PCR data with duplicated measurements are shown. (D) WT or BATF KO CD4 + T cells were activated for 4–5 d with anti-CD3/CD28 in the presence of IL-2 and 10 nM RA and transfected with pGL4-5′-Itg-α4. The cells were reactivated with anti-CD3/28 + IL2 in the presence of 20 nM RA for 16 h, and luciferase activity was normalized to control Renilla luciferase activity. (E) Naive WT or BATF KO CD4 + T cells were activated with anti-CD3/CD28 or concanavalin A for 4–5 d in the presence of IL-2 and 10 nM RA and the indicated HDAC inhibitors (TSA, BML-210, or EX-527), and the expression of CCR9 and α4β7 was measured by flow cytometry. Graphs show the percentage of cells expressing CCR9 or α4β7. All of the experiments were performed at least three times, and pooled (A, D, and E) or representative (B and C) data are shown. Error bars are SEM obtained from pooled data (A, D, and E) or differences between duplicated measurements (B and C). Significant differences from the KO control group are shown (*, P
Figure Legend Snippet: RARα fails to bind the 5′ regulatory regions of the mouse CCR9 and Itg-α4 genes in BATF deficiency. WT or BATF KO CD4 + T cells were activated for 4–5 d with concanavalin A, IL-2, and RA. (A) Expression of nuclear RAR genes in BATF KO CD4 + T cells was examined by quantitative RT-PCR. Normalized values to β-actin levels are shown. (B and C) The binding of RARα and BATF and histone H4 acetylation on the CCR9 gene (B) or the Itg-α4 gene (C) were assessed by ChIP assay. Representative PCR data with duplicated measurements are shown. (D) WT or BATF KO CD4 + T cells were activated for 4–5 d with anti-CD3/CD28 in the presence of IL-2 and 10 nM RA and transfected with pGL4-5′-Itg-α4. The cells were reactivated with anti-CD3/28 + IL2 in the presence of 20 nM RA for 16 h, and luciferase activity was normalized to control Renilla luciferase activity. (E) Naive WT or BATF KO CD4 + T cells were activated with anti-CD3/CD28 or concanavalin A for 4–5 d in the presence of IL-2 and 10 nM RA and the indicated HDAC inhibitors (TSA, BML-210, or EX-527), and the expression of CCR9 and α4β7 was measured by flow cytometry. Graphs show the percentage of cells expressing CCR9 or α4β7. All of the experiments were performed at least three times, and pooled (A, D, and E) or representative (B and C) data are shown. Error bars are SEM obtained from pooled data (A, D, and E) or differences between duplicated measurements (B and C). Significant differences from the KO control group are shown (*, P

Techniques Used: Expressing, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay, Flow Cytometry, Cytometry

11) Product Images from "Kr?ppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor ?-mediated, Not by Retinoic Acid Receptor ?-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells *"

Article Title: Kr?ppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor ?-mediated, Not by Retinoic Acid Receptor ?-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.026989

Klf4 down-regulates the expression and activity of RARα. A , immediately following balloon injury, vehicle, pAd, or pAd-Klf4 was infused into the segment of the common carotid artery as described under “Experimental Procedures.”
Figure Legend Snippet: Klf4 down-regulates the expression and activity of RARα. A , immediately following balloon injury, vehicle, pAd, or pAd-Klf4 was infused into the segment of the common carotid artery as described under “Experimental Procedures.”

Techniques Used: Expressing, Activity Assay

Effects of Klf4 on RARα-mediated increase in phosphoactive Akt and ERK levels. A , VSMCs were infected with pAd, pAd-Klf4, or pAd-Klf4 plus pCMV-hRARα for 48 h. Serum-starved cells were incubated with vehicle (DMSO) or the RARα
Figure Legend Snippet: Effects of Klf4 on RARα-mediated increase in phosphoactive Akt and ERK levels. A , VSMCs were infected with pAd, pAd-Klf4, or pAd-Klf4 plus pCMV-hRARα for 48 h. Serum-starved cells were incubated with vehicle (DMSO) or the RARα

Techniques Used: Infection, Incubation

Klf4-induced growth arrest is not dependent on RARα. A , Am80 or Ro 41-5253 was given orally to pAd- and pAd-Klf4-infected animals that were subjected or not to vascular injury. Three days later, the injured arteries were harvested and analyzed
Figure Legend Snippet: Klf4-induced growth arrest is not dependent on RARα. A , Am80 or Ro 41-5253 was given orally to pAd- and pAd-Klf4-infected animals that were subjected or not to vascular injury. Three days later, the injured arteries were harvested and analyzed

Techniques Used: Infection

12) Product Images from "Sulindac Sulfide Reverses Aberrant Self-Renewal of Progenitor Cells Induced by the AML-Associated Fusion Proteins PML/RAR? and PLZF/RAR?"

Article Title: Sulindac Sulfide Reverses Aberrant Self-Renewal of Progenitor Cells Induced by the AML-Associated Fusion Proteins PML/RAR? and PLZF/RAR?

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022540

Effect of SSi on the biology of X-RARα-, γ-catenin- and constitutively active β-catenin-expressing murine HSCs. ( A ) Experimental strategy. Sca1 + /lin - BM cells were infected with the indicated retroviruses and plated in semi-solid medium with the indicated growth factors to determine the serial plating potential in the presence of either 100 µM SSi or 0.02% DMSO. ( B ) Long-term serial replating (I–VII) of HSC and X-RARα-positive HSC. SSi withdrawal after plating round V for PML/RARα-positive colonies and after plating round VI for PLZF/RARα-positive colonies. ( C ) Colony morphology of plating rounds I, III, VI, and VII as well as SSi withdrawal. ( D ) Long-term serial replating (I–V) of γ-catenin- and β-catenin-S33A-positive HSC. Data are expressed as the mean of three independent experiments with SD. Control - empty vector; ↓-SSi withdrawal; I–VII: serial plating rounds.
Figure Legend Snippet: Effect of SSi on the biology of X-RARα-, γ-catenin- and constitutively active β-catenin-expressing murine HSCs. ( A ) Experimental strategy. Sca1 + /lin - BM cells were infected with the indicated retroviruses and plated in semi-solid medium with the indicated growth factors to determine the serial plating potential in the presence of either 100 µM SSi or 0.02% DMSO. ( B ) Long-term serial replating (I–VII) of HSC and X-RARα-positive HSC. SSi withdrawal after plating round V for PML/RARα-positive colonies and after plating round VI for PLZF/RARα-positive colonies. ( C ) Colony morphology of plating rounds I, III, VI, and VII as well as SSi withdrawal. ( D ) Long-term serial replating (I–V) of γ-catenin- and β-catenin-S33A-positive HSC. Data are expressed as the mean of three independent experiments with SD. Control - empty vector; ↓-SSi withdrawal; I–VII: serial plating rounds.

Techniques Used: Expressing, Infection, Plasmid Preparation

Effect of SSi on the X-RARα-induced phenotype in murine HSCs. ( A ) Experimental strategy. Sca1+/lin - BM cells were infected with the indicated retroviruses and maintained for one week in liquid culture with the indicated growth factors in the presence/absence of 40 µM SSi. ( B ) Differentiation was assessed by the expression of Gr-1, Mac-1, c-Kit and Sca1. ( C ) Schematic representation of the proliferation competition assay (PCA). ( D ) GFP expression in X-RARα-positive HPCs assessed by FACS analysis in the presence/absence of SSi at day 2, 4, and 7. One representative result from three independent experiments is shown.
Figure Legend Snippet: Effect of SSi on the X-RARα-induced phenotype in murine HSCs. ( A ) Experimental strategy. Sca1+/lin - BM cells were infected with the indicated retroviruses and maintained for one week in liquid culture with the indicated growth factors in the presence/absence of 40 µM SSi. ( B ) Differentiation was assessed by the expression of Gr-1, Mac-1, c-Kit and Sca1. ( C ) Schematic representation of the proliferation competition assay (PCA). ( D ) GFP expression in X-RARα-positive HPCs assessed by FACS analysis in the presence/absence of SSi at day 2, 4, and 7. One representative result from three independent experiments is shown.

Techniques Used: Infection, Expressing, Competitive Binding Assay, FACS

Effect of SSi on PML/RARα-mediated aberrant long-term stem cell capacity in murine HSC. ( A ) Experimental strategy for studying the LT-HSC capacity of PML/RARα-expressing HSCs. Sca1 + /lin - BM cells from CD45.1 + mice were infected with the indicated retroviruses and maintained for one week in liquid culture with the indicated growth factors in the presence/absence of 40 µM SSi. The cells were co-transplanted with CD45.2 + BM cells into lethally (11 Gy) irradiated CD45.2 + recipient mice. ( B ) Donor chimerism 8 months after transplantation was used to determine LT-HSC capacity in the BM and spleen. Statistical significance was determined using the Log-rank (Mantel-Cox) test (*
Figure Legend Snippet: Effect of SSi on PML/RARα-mediated aberrant long-term stem cell capacity in murine HSC. ( A ) Experimental strategy for studying the LT-HSC capacity of PML/RARα-expressing HSCs. Sca1 + /lin - BM cells from CD45.1 + mice were infected with the indicated retroviruses and maintained for one week in liquid culture with the indicated growth factors in the presence/absence of 40 µM SSi. The cells were co-transplanted with CD45.2 + BM cells into lethally (11 Gy) irradiated CD45.2 + recipient mice. ( B ) Donor chimerism 8 months after transplantation was used to determine LT-HSC capacity in the BM and spleen. Statistical significance was determined using the Log-rank (Mantel-Cox) test (*

Techniques Used: Expressing, Mouse Assay, Infection, Irradiation, Transplantation Assay

Induction of apoptosis in patient-derived NB4 cells by Sulindac derivatives. Rate of apoptosis in patient-derived PML/RARα-positive NB4 cells was assessed by 7AAD staining upon exposure to clinically achievable concentrations (50–150 µM) of SSi and SSo. Apoptosis was measured after 72 h. Data are expressed as the mean of three independent experiments with standard deviation (SD). Statistical analysis was performed using Student's t -test (* - p
Figure Legend Snippet: Induction of apoptosis in patient-derived NB4 cells by Sulindac derivatives. Rate of apoptosis in patient-derived PML/RARα-positive NB4 cells was assessed by 7AAD staining upon exposure to clinically achievable concentrations (50–150 µM) of SSi and SSo. Apoptosis was measured after 72 h. Data are expressed as the mean of three independent experiments with standard deviation (SD). Statistical analysis was performed using Student's t -test (* - p

Techniques Used: Derivative Assay, Staining, Standard Deviation

Effect of SSi on PML/RARα-mediated Wnt signaling pathway activation. ( A ) Transactivation assay for Wnt-signaling-related TCF/LEF-dependent transcription. Indicated expression vectors were co-transfected with either Topflash (OT) or Fopflash (OF) (the pGL3-OT promoter contains three TCF/LEF binding sites, whereas pGL3-OF contains mutated inactive binding sites and is a negative control) reporter constructs into 293 cells and exposed them to either 100 µM SSi or 0.02% DMSO. After 48 h, luciferase activity was measured and normalized to co-transfected Renilla activity. ( B ) Western blots for the expression of PML/RARα and S33A using the indicated antibodies, α-β-actin-loading control. ( C ) Effects of SSi on Wnt target genes in X-RARα-positive Sca1 + /lin - HPCs. Results are represented as 2 -ΔΔCT values. Each experiment was performed three times in triplicate, with similar results obtained each time. One representative experiment with SD is shown.
Figure Legend Snippet: Effect of SSi on PML/RARα-mediated Wnt signaling pathway activation. ( A ) Transactivation assay for Wnt-signaling-related TCF/LEF-dependent transcription. Indicated expression vectors were co-transfected with either Topflash (OT) or Fopflash (OF) (the pGL3-OT promoter contains three TCF/LEF binding sites, whereas pGL3-OF contains mutated inactive binding sites and is a negative control) reporter constructs into 293 cells and exposed them to either 100 µM SSi or 0.02% DMSO. After 48 h, luciferase activity was measured and normalized to co-transfected Renilla activity. ( B ) Western blots for the expression of PML/RARα and S33A using the indicated antibodies, α-β-actin-loading control. ( C ) Effects of SSi on Wnt target genes in X-RARα-positive Sca1 + /lin - HPCs. Results are represented as 2 -ΔΔCT values. Each experiment was performed three times in triplicate, with similar results obtained each time. One representative experiment with SD is shown.

Techniques Used: Activation Assay, Transactivation Assay, Expressing, Transfection, Binding Assay, Negative Control, Construct, Luciferase, Activity Assay, Western Blot

The effect of Sulindac derivatives on the expression of β-catenin and γ-catenin in PML/RARα-positive leukemic cells. Protein expression of active β-catenin, total β-catenin, γ-catenin and PML/RARα was assessed by western blot analysis with the indicated antibodies. ( A ) Patient-derived PML/RARα-positive NB4 cells treated with SSo (200 µM), SSi (200 µM) or 0.02% DMSO at 24 h and 48 h. ( B ) PML/RARα-expressing KG-1 cells treated at the cell type-specific IC 50 (100 µM - SSi, 360 µM - SSo and 0.02% DMSO) at 72 h. Control - empty-vector-transfected cells; α-α-tubulin-loading control. ( C – D ) protein quantification of western blots presented in panel A and B, the bars represent the band intensity relative to the intensity of the tubulin bands reveled with a S-800 Densitometer and the Quantity One software (Bio-Rad).
Figure Legend Snippet: The effect of Sulindac derivatives on the expression of β-catenin and γ-catenin in PML/RARα-positive leukemic cells. Protein expression of active β-catenin, total β-catenin, γ-catenin and PML/RARα was assessed by western blot analysis with the indicated antibodies. ( A ) Patient-derived PML/RARα-positive NB4 cells treated with SSo (200 µM), SSi (200 µM) or 0.02% DMSO at 24 h and 48 h. ( B ) PML/RARα-expressing KG-1 cells treated at the cell type-specific IC 50 (100 µM - SSi, 360 µM - SSo and 0.02% DMSO) at 72 h. Control - empty-vector-transfected cells; α-α-tubulin-loading control. ( C – D ) protein quantification of western blots presented in panel A and B, the bars represent the band intensity relative to the intensity of the tubulin bands reveled with a S-800 Densitometer and the Quantity One software (Bio-Rad).

Techniques Used: Expressing, Western Blot, Derivative Assay, Plasmid Preparation, Transfection, Software

Effect of SSi on X-RARα-mediated aberrant stem cell capacity in murine HSC. ( A ) Experimental strategy for studying the ST-HSC capacity of X-RARα-expressing HSCs (CFU-S12). Sca1 + /lin - BM cells were infected with the indicated retroviruses and maintained for one week in liquid culture with the indicated growth factors in the presence/absence of 40 µM SSi. Cells were inoculated into lethally irradiated (11Gy) recipients that were then sacrificed at day 12 after transplantation. ( B ) Photographs of all the fixed spleen are shown, the arrows indicate an example of a CFU in the fixed spleens. ( C ) Bulks represent the number of CFU-S12 expressed as the mean from three spleens with SD. Statistical analysis was performed using Student's t -test (* - p
Figure Legend Snippet: Effect of SSi on X-RARα-mediated aberrant stem cell capacity in murine HSC. ( A ) Experimental strategy for studying the ST-HSC capacity of X-RARα-expressing HSCs (CFU-S12). Sca1 + /lin - BM cells were infected with the indicated retroviruses and maintained for one week in liquid culture with the indicated growth factors in the presence/absence of 40 µM SSi. Cells were inoculated into lethally irradiated (11Gy) recipients that were then sacrificed at day 12 after transplantation. ( B ) Photographs of all the fixed spleen are shown, the arrows indicate an example of a CFU in the fixed spleens. ( C ) Bulks represent the number of CFU-S12 expressed as the mean from three spleens with SD. Statistical analysis was performed using Student's t -test (* - p

Techniques Used: Expressing, Infection, Irradiation, Transplantation Assay

13) Product Images from "The Roles of GSK-3β in Regulation of Retinoid Signaling and Sorafenib Treatment Response in Hepatocellular Carcinoma"

Article Title: The Roles of GSK-3β in Regulation of Retinoid Signaling and Sorafenib Treatment Response in Hepatocellular Carcinoma

Journal: Theranostics

doi: 10.7150/thno.38711

GSK-3β impairs retinoid signaling. (A) Reporter assays. HepG2 and HepG2/3β cells were transfected with myc-RXRα or myc-RXRα/S78A. The cells were co-transfected with pGL6-βRARE, Renilla, and HA-RARα for 24 h and then treated vehicle or 1 μM 9- cis -RA for 20 h. The fluorescence intensities were determined with Multiskan Spectrum (PerkinElmer). Renilla luciferase activity was used to normalize for transfection efficiency. ** p
Figure Legend Snippet: GSK-3β impairs retinoid signaling. (A) Reporter assays. HepG2 and HepG2/3β cells were transfected with myc-RXRα or myc-RXRα/S78A. The cells were co-transfected with pGL6-βRARE, Renilla, and HA-RARα for 24 h and then treated vehicle or 1 μM 9- cis -RA for 20 h. The fluorescence intensities were determined with Multiskan Spectrum (PerkinElmer). Renilla luciferase activity was used to normalize for transfection efficiency. ** p

Techniques Used: Transfection, Fluorescence, Luciferase, Activity Assay

14) Product Images from "SP1 and RARα regulate AGAP2 expression in cancer"

Article Title: SP1 and RARα regulate AGAP2 expression in cancer

Journal: Scientific Reports

doi: 10.1038/s41598-018-36888-x

Mechanism of ATRA-mediated AGAP2 transcription. ( a ) Sheared chromatin of DU145 cells grown under AGAP2 expression conditions was used for immunoprecipitation using 2 μg of antibody (a rabbit IgG as negative control, a rabbit antibody against RNApol II as positive control, a rabbit anti-RARα, a rabbit anti-RXRα or a rabbit anti-PCAF antibody) and optimised primers were used to amplify a region specific to the AGAP2 ). Data are represented as fold enrichment: fold enrich in signal relative to the IgG background signal. ( b ) Diagram representing the proposed mechanism of ATRA-mediated activation of AGAP2 transcription. In the presence of ATRA (lower panel), the heterodimer RARα/RXRα would recruit the lysine acetyl transferase PCAF to activate transcription and the recruitment of SP1 would also be enhanced.
Figure Legend Snippet: Mechanism of ATRA-mediated AGAP2 transcription. ( a ) Sheared chromatin of DU145 cells grown under AGAP2 expression conditions was used for immunoprecipitation using 2 μg of antibody (a rabbit IgG as negative control, a rabbit antibody against RNApol II as positive control, a rabbit anti-RARα, a rabbit anti-RXRα or a rabbit anti-PCAF antibody) and optimised primers were used to amplify a region specific to the AGAP2 ). Data are represented as fold enrichment: fold enrich in signal relative to the IgG background signal. ( b ) Diagram representing the proposed mechanism of ATRA-mediated activation of AGAP2 transcription. In the presence of ATRA (lower panel), the heterodimer RARα/RXRα would recruit the lysine acetyl transferase PCAF to activate transcription and the recruitment of SP1 would also be enhanced.

Techniques Used: Expressing, Immunoprecipitation, Negative Control, Positive Control, Activation Assay

15) Product Images from "SP1 and RARα regulate AGAP2 expression in cancer"

Article Title: SP1 and RARα regulate AGAP2 expression in cancer

Journal: Scientific Reports

doi: 10.1038/s41598-018-36888-x

Mechanism of ATRA-mediated AGAP2 transcription. ( a ) Sheared chromatin of DU145 cells grown under AGAP2 expression conditions was used for immunoprecipitation using 2 μg of antibody (a rabbit IgG as negative control, a rabbit antibody against RNApol II as positive control, a rabbit anti-RARα, a rabbit anti-RXRα or a rabbit anti-PCAF antibody) and optimised primers were used to amplify a region specific to the AGAP2 promoter (see Supplementary Table 1 ). Data are represented as fold enrichment: fold enrich in signal relative to the IgG background signal. ( b ) Diagram representing the proposed mechanism of ATRA-mediated activation of AGAP2 transcription. In the presence of ATRA (lower panel), the heterodimer RARα/RXRα would recruit the lysine acetyl transferase PCAF to activate transcription and the recruitment of SP1 would also be enhanced.
Figure Legend Snippet: Mechanism of ATRA-mediated AGAP2 transcription. ( a ) Sheared chromatin of DU145 cells grown under AGAP2 expression conditions was used for immunoprecipitation using 2 μg of antibody (a rabbit IgG as negative control, a rabbit antibody against RNApol II as positive control, a rabbit anti-RARα, a rabbit anti-RXRα or a rabbit anti-PCAF antibody) and optimised primers were used to amplify a region specific to the AGAP2 promoter (see Supplementary Table 1 ). Data are represented as fold enrichment: fold enrich in signal relative to the IgG background signal. ( b ) Diagram representing the proposed mechanism of ATRA-mediated activation of AGAP2 transcription. In the presence of ATRA (lower panel), the heterodimer RARα/RXRα would recruit the lysine acetyl transferase PCAF to activate transcription and the recruitment of SP1 would also be enhanced.

Techniques Used: Expressing, Immunoprecipitation, Negative Control, Positive Control, Activation Assay

16) Product Images from "Retinoic acid regulates cell cycle progression and cell differentiation in human monocytic THP-1 cells"

Article Title: Retinoic acid regulates cell cycle progression and cell differentiation in human monocytic THP-1 cells

Journal: Experimental cell research

doi: 10.1016/j.yexcr.2004.02.017

Regulation of cyclin E gene expression in THP-1 cells by retinoic acid. THP-1 cells were plated in RPMI 1640 medium with 5% serum for 24 h and then with 2% FBS for another 24 h. Serum was added to cells at a final concentration of 10% and RA was added to cells at the indicated concentrations. Total RNA was isolated at the end of each treatment and subjected to RT-PCR analysis. (A) Expression level of the cyclin E gene in the absence or presence of RA at different concentrations for 24 h. The level is represented by the ratio of treated cells over the control without RA. (B) Expression of cyclin E in the presence of RA for different times. The numbers shown in graphs A and B are the mean ± SD of three independent experiments. (C) Western blot analysis showing the levels of cyclin E in the presence (10 nM) and absence of RA. The image represents one of three independent experiments performed in duplicate. Same samples were also subjected to the Western blot analysis with anti-actin antibody and the result is shown underneath. (D) Western blot analysis of the same THP-1 cell protein lysates as described in (C) using antibodies specific to RXRα and RARα. (E) Graph showing the correlation between the protein levels of cyclin E and RXRα, both regulated by RA. The axes are arbitrary density units. (F) Western blot analysis of p27 protein in the presence and absence of RA. The actin bands were shown underneath as internal control.
Figure Legend Snippet: Regulation of cyclin E gene expression in THP-1 cells by retinoic acid. THP-1 cells were plated in RPMI 1640 medium with 5% serum for 24 h and then with 2% FBS for another 24 h. Serum was added to cells at a final concentration of 10% and RA was added to cells at the indicated concentrations. Total RNA was isolated at the end of each treatment and subjected to RT-PCR analysis. (A) Expression level of the cyclin E gene in the absence or presence of RA at different concentrations for 24 h. The level is represented by the ratio of treated cells over the control without RA. (B) Expression of cyclin E in the presence of RA for different times. The numbers shown in graphs A and B are the mean ± SD of three independent experiments. (C) Western blot analysis showing the levels of cyclin E in the presence (10 nM) and absence of RA. The image represents one of three independent experiments performed in duplicate. Same samples were also subjected to the Western blot analysis with anti-actin antibody and the result is shown underneath. (D) Western blot analysis of the same THP-1 cell protein lysates as described in (C) using antibodies specific to RXRα and RARα. (E) Graph showing the correlation between the protein levels of cyclin E and RXRα, both regulated by RA. The axes are arbitrary density units. (F) Western blot analysis of p27 protein in the presence and absence of RA. The actin bands were shown underneath as internal control.

Techniques Used: Expressing, Concentration Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

17) Product Images from "Retinoic Acid Repression of Bone Morphogenetic Protein 4 in Inner Ear Development"

Article Title: Retinoic Acid Repression of Bone Morphogenetic Protein 4 in Inner Ear Development

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.7.2277-2286.2003

RAR expression. (A) Analysis of RARα and RARγ expression in 2B1 cells by Western blot analysis. (B) Schematic diagram of a prototypical RAR. Three RAR genes (α, β, and γ) encode receptors that are unrelated in their amino-terminal A and B domains but highly similar in their DNA and ligand binding domains (DBD and LBD, respectively).
Figure Legend Snippet: RAR expression. (A) Analysis of RARα and RARγ expression in 2B1 cells by Western blot analysis. (B) Schematic diagram of a prototypical RAR. Three RAR genes (α, β, and γ) encode receptors that are unrelated in their amino-terminal A and B domains but highly similar in their DNA and ligand binding domains (DBD and LBD, respectively).

Techniques Used: Expressing, Western Blot, Ligand Binding Assay

18) Product Images from "Inhibition of long non-coding RNA NEAT1 impairs myeloid differentiation in acute promyelocytic leukemia cells"

Article Title: Inhibition of long non-coding RNA NEAT1 impairs myeloid differentiation in acute promyelocytic leukemia cells

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-693

lncRNA NEAT1 is repressed in cells expressing PML-RARα and upregulated in response to ATRA. (A) qRT–PCR analysis of NEAT1 in U937-PR9 cells treated with 100 μM ZnSO 4 at the indicated time points. A time series of induction for the PML-RARα protein by ZnSO 4 is shown in Additional file 2 : Figure S2A. (B) qRT–PCR analysis of NEAT1 after knocking down PML-RARα. (C) NB4 and NB4-R2 cells were treated with 1 μM ATRA. NEAT1 was measured by qRT-PCR and normalized to the housekeeping gene ACTB . The panels show the mean ± SD of a representative experiment performed in triplicate.
Figure Legend Snippet: lncRNA NEAT1 is repressed in cells expressing PML-RARα and upregulated in response to ATRA. (A) qRT–PCR analysis of NEAT1 in U937-PR9 cells treated with 100 μM ZnSO 4 at the indicated time points. A time series of induction for the PML-RARα protein by ZnSO 4 is shown in Additional file 2 : Figure S2A. (B) qRT–PCR analysis of NEAT1 after knocking down PML-RARα. (C) NB4 and NB4-R2 cells were treated with 1 μM ATRA. NEAT1 was measured by qRT-PCR and normalized to the housekeeping gene ACTB . The panels show the mean ± SD of a representative experiment performed in triplicate.

Techniques Used: Expressing, Quantitative RT-PCR

19) Product Images from "A phosphorylation defective retinoic acid receptor mutant mimics the effects of retinoic acid on EGFR mediated AP-1 expression and cancer cell proliferation"

Article Title: A phosphorylation defective retinoic acid receptor mutant mimics the effects of retinoic acid on EGFR mediated AP-1 expression and cancer cell proliferation

Journal: Cancer Cell International

doi: 10.1186/1475-2867-2-15

The phosphorylation defective RARα mutant inhibits G1 to S phase progression in human cancer cells. Decreased BrdU incorporation by carcinoma cells expressing the phosphorylation defective RARα mutant. Cultures of G418 resistant (neo), HA tagged RARα (HA1,2), and S77A RARα mutant (RAR1-4) SCC25 clones were subjected to BrdU incorporation analysis as described in Methods. The number of BrdU positive cells was expressed as a percentage of total cells counted in ten high power fields. These experiments were performed three times with similar results. Error bars indicate SEM.
Figure Legend Snippet: The phosphorylation defective RARα mutant inhibits G1 to S phase progression in human cancer cells. Decreased BrdU incorporation by carcinoma cells expressing the phosphorylation defective RARα mutant. Cultures of G418 resistant (neo), HA tagged RARα (HA1,2), and S77A RARα mutant (RAR1-4) SCC25 clones were subjected to BrdU incorporation analysis as described in Methods. The number of BrdU positive cells was expressed as a percentage of total cells counted in ten high power fields. These experiments were performed three times with similar results. Error bars indicate SEM.

Techniques Used: Mutagenesis, BrdU Incorporation Assay, Expressing, Clone Assay

The phosphorylation defective RARα mutant inhibits EGFR expression, ERK1 activation, and AP-1 protein levels. (A) Cultures of G418 resistant (neo) and S77A RARα mutant (RAR1-4) SCC25 clones were harvested for western blot as described in Methods. Blots were incubated with anti-human primary antibodies to AP-1 proteins Fra-1, c-jun, or JunB. These experiments were performed three times using different lysates with similar results. Representative blots are shown.
Figure Legend Snippet: The phosphorylation defective RARα mutant inhibits EGFR expression, ERK1 activation, and AP-1 protein levels. (A) Cultures of G418 resistant (neo) and S77A RARα mutant (RAR1-4) SCC25 clones were harvested for western blot as described in Methods. Blots were incubated with anti-human primary antibodies to AP-1 proteins Fra-1, c-jun, or JunB. These experiments were performed three times using different lysates with similar results. Representative blots are shown.

Techniques Used: Mutagenesis, Expressing, Activation Assay, Clone Assay, Western Blot, Incubation

The phosphorylation defective RARα mutant inhibits AP-1 activity in the absence of RA. Triplicate cultures of SCC25 cells were transiently transfected with a heterologous promoter construct containing an AP-1 response element in the luciferase reporter vector pGL3 as described in Methods. Wild type (alpha) or S77A mutant RARα expression vectors were cotransfected with the reporter constructs. Blank expression vector was used to control for the amount of DNA transfected. Transfected cultures treated with vehicle or 1 μM RA for 24 hours were used as controls for AP-1 inhibition. Relative light units from vehicle treated cells transfected with reporter construct and blank vector were assigned an arbitrary AP-1 activity value of 100. These experiments were performed three times with similar results. Error bars indicate SEM.
Figure Legend Snippet: The phosphorylation defective RARα mutant inhibits AP-1 activity in the absence of RA. Triplicate cultures of SCC25 cells were transiently transfected with a heterologous promoter construct containing an AP-1 response element in the luciferase reporter vector pGL3 as described in Methods. Wild type (alpha) or S77A mutant RARα expression vectors were cotransfected with the reporter constructs. Blank expression vector was used to control for the amount of DNA transfected. Transfected cultures treated with vehicle or 1 μM RA for 24 hours were used as controls for AP-1 inhibition. Relative light units from vehicle treated cells transfected with reporter construct and blank vector were assigned an arbitrary AP-1 activity value of 100. These experiments were performed three times with similar results. Error bars indicate SEM.

Techniques Used: Mutagenesis, Activity Assay, Transfection, Construct, Luciferase, Plasmid Preparation, Expressing, Inhibition

EGFR expression and ERK1 activation are inhibited in S77A RARα mutant cells. Cells were harvested for western blot as described above. Blots were incubated with anti-human primary antibodies to EGFR, activated ERK1 (anti-pERK1), and total ERK1. These experiments were performed three times using different lysates with similar results. Representative blots are shown.
Figure Legend Snippet: EGFR expression and ERK1 activation are inhibited in S77A RARα mutant cells. Cells were harvested for western blot as described above. Blots were incubated with anti-human primary antibodies to EGFR, activated ERK1 (anti-pERK1), and total ERK1. These experiments were performed three times using different lysates with similar results. Representative blots are shown.

Techniques Used: Expressing, Activation Assay, Mutagenesis, Western Blot, Incubation

The phosphorylation defective RARα mutant inhibits proliferation of human cancer cells. Expression of the S77A RARα mutant decreased endogenous RARα phosphorylation in SCC clones (RAR1-4) compared to G418 resistant control cells (neo). RARα protein was immunoprecipitated as described above.
Figure Legend Snippet: The phosphorylation defective RARα mutant inhibits proliferation of human cancer cells. Expression of the S77A RARα mutant decreased endogenous RARα phosphorylation in SCC clones (RAR1-4) compared to G418 resistant control cells (neo). RARα protein was immunoprecipitated as described above.

Techniques Used: Mutagenesis, Expressing, Clone Assay, Immunoprecipitation

The S77A mutant is not phosphorylated on serine in SCC25 cells. Cells were stably transfected with HA tagged RARα (HA-RAR1,2), S77A mutant (HA-S77A1,2), or G418 resistance (neo) expression vectors. HA tagged RARα proteins were immunoprecipitated from cellular lysates using anti-HA antibody (IP HA). Blots were incubated with anti-phosphoserine antibodies (anti-pSer) to determine relative levels of RARα phosphorylation on serine residues. Blots were stripped and incubated with anti-RARα antibody to confirm the identity of the anti-HA immunoprecipitates. Blots were incubated with anti-HA antibody to ensure equal amounts of immunoprecipitated protein in each lane. These experiments were performed three times using different clones with similar results. Representative blots are shown.
Figure Legend Snippet: The S77A mutant is not phosphorylated on serine in SCC25 cells. Cells were stably transfected with HA tagged RARα (HA-RAR1,2), S77A mutant (HA-S77A1,2), or G418 resistance (neo) expression vectors. HA tagged RARα proteins were immunoprecipitated from cellular lysates using anti-HA antibody (IP HA). Blots were incubated with anti-phosphoserine antibodies (anti-pSer) to determine relative levels of RARα phosphorylation on serine residues. Blots were stripped and incubated with anti-RARα antibody to confirm the identity of the anti-HA immunoprecipitates. Blots were incubated with anti-HA antibody to ensure equal amounts of immunoprecipitated protein in each lane. These experiments were performed three times using different clones with similar results. Representative blots are shown.

Techniques Used: Mutagenesis, Stable Transfection, Transfection, Expressing, Immunoprecipitation, Incubation, Clone Assay

The S77A RARα mutant inhibits proliferation of SCC25 cells. G418 resistant (neo), wild type HA-tagged RARα (HA1,2), and S77A mutant clones (RAR1-4) were cultured as described in Methods. At two day intervals, the cells in triplicate cultures were counted with a hemacytometer. These experiments were performed three times with similar results. Error bars indicate SEM.
Figure Legend Snippet: The S77A RARα mutant inhibits proliferation of SCC25 cells. G418 resistant (neo), wild type HA-tagged RARα (HA1,2), and S77A mutant clones (RAR1-4) were cultured as described in Methods. At two day intervals, the cells in triplicate cultures were counted with a hemacytometer. These experiments were performed three times with similar results. Error bars indicate SEM.

Techniques Used: Mutagenesis, Clone Assay, Cell Culture

Cultures of G418 resistant (neo) and S77A RARα mutant (RAR1-4) SCC25 clones were harvested for western blot as described in Methods. Blots were incubated with primary antibodies to the G1 cyclin dependent kinase inhibitors p21 WAF1/Cip1 and p27 Kip1 and to the S phase cyclin A. These experiments were performed three times using different lysates with similar results. Representative blots are shown.
Figure Legend Snippet: Cultures of G418 resistant (neo) and S77A RARα mutant (RAR1-4) SCC25 clones were harvested for western blot as described in Methods. Blots were incubated with primary antibodies to the G1 cyclin dependent kinase inhibitors p21 WAF1/Cip1 and p27 Kip1 and to the S phase cyclin A. These experiments were performed three times using different lysates with similar results. Representative blots are shown.

Techniques Used: Mutagenesis, Clone Assay, Western Blot, Incubation

RA decreases cyclin H-cdk7 expression and inhibits RARα phosphorylation. (A) SCC4 and SCC25 cells were treated with vehicle or 1 μM RA (+RA) for 16 hours as described in Methods. Cyclin H and cdk7 expression was determined by western blot. Blots were stripped and incubated with anti-β-actin antibody to ensure equal amounts of protein in each lane. (B) RA inhibits RARα phosphorylation. RARα was immunoprecipitated (IP RARα) from lysates of vehicle and RA treated SCC4 and SCC25 cells as described in Methods. Blots were incubated with anti-phosphoserine antibodies (anti-pSer) followed by stripping and probing with anti-RARα antibodies to ensure equal relative amounts of immunoprecipitated protein in each lane. To determine the specificity of this assay, SCC25 cells were treated with vehicle or the selective MAPK inhibitors PD98059 (50 μM) or SB203580 (10 μM) for up to 24 hours. RARα protein was immunoprecipitated as described above. These experiments were performed three times with similar results. Representative blots are shown.
Figure Legend Snippet: RA decreases cyclin H-cdk7 expression and inhibits RARα phosphorylation. (A) SCC4 and SCC25 cells were treated with vehicle or 1 μM RA (+RA) for 16 hours as described in Methods. Cyclin H and cdk7 expression was determined by western blot. Blots were stripped and incubated with anti-β-actin antibody to ensure equal amounts of protein in each lane. (B) RA inhibits RARα phosphorylation. RARα was immunoprecipitated (IP RARα) from lysates of vehicle and RA treated SCC4 and SCC25 cells as described in Methods. Blots were incubated with anti-phosphoserine antibodies (anti-pSer) followed by stripping and probing with anti-RARα antibodies to ensure equal relative amounts of immunoprecipitated protein in each lane. To determine the specificity of this assay, SCC25 cells were treated with vehicle or the selective MAPK inhibitors PD98059 (50 μM) or SB203580 (10 μM) for up to 24 hours. RARα protein was immunoprecipitated as described above. These experiments were performed three times with similar results. Representative blots are shown.

Techniques Used: Expressing, Western Blot, Incubation, Immunoprecipitation, Stripping Membranes

20) Product Images from "High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis"

Article Title: High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis

Journal: Genes & Cancer

doi:

CRBP-1 transfection influences transcriptional pathways and differentiation of A549 adenocarcinoma cells A, bar graph after real-time PCR showing keratin (K) 1, 5, 14 and involucrin up-regulation, K10 down-regulation and unmodified K7 transcription level in CRBP-1 + compared to CRBP-1 − A549 cells. B, densitometric analysis of RARα, RARβ and RXRα protein expression by blot analysis. C, bar graph of RARs, RXRα, PPARβ/δ, FABP5 and CRABP-2 transcripts. D, bar graph and E, heat map of RT 2 profiler TM PCR assay of EGF/PDGF signaling-specific genes in CRBP-1 + A549 cells. Up-regulated and down-regulated genes are in dark grey and light grey, respectively. F, densitometric analysis of creb1, c-jun, p53, pAkt/Akt, pEGFR/EGFR and pErk1/2 protein expression by blots. Columns are means ± SEM of three different experiments. * p
Figure Legend Snippet: CRBP-1 transfection influences transcriptional pathways and differentiation of A549 adenocarcinoma cells A, bar graph after real-time PCR showing keratin (K) 1, 5, 14 and involucrin up-regulation, K10 down-regulation and unmodified K7 transcription level in CRBP-1 + compared to CRBP-1 − A549 cells. B, densitometric analysis of RARα, RARβ and RXRα protein expression by blot analysis. C, bar graph of RARs, RXRα, PPARβ/δ, FABP5 and CRABP-2 transcripts. D, bar graph and E, heat map of RT 2 profiler TM PCR assay of EGF/PDGF signaling-specific genes in CRBP-1 + A549 cells. Up-regulated and down-regulated genes are in dark grey and light grey, respectively. F, densitometric analysis of creb1, c-jun, p53, pAkt/Akt, pEGFR/EGFR and pErk1/2 protein expression by blots. Columns are means ± SEM of three different experiments. * p

Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

21) Product Images from "p27Kip1 deficiency promotes prostate carcinogenesis but does not affect the efficacy of retinoids in suppressing the neoplastic process"

Article Title: p27Kip1 deficiency promotes prostate carcinogenesis but does not affect the efficacy of retinoids in suppressing the neoplastic process

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-541

Morphological changes in DLP of p27 deficient mice in the course of carcinogenesis . 1A: Dorso-lateral prostate of 6-month-old p27-/- mice. Note the glandular structures are covered by a single layer of prostate epithelial cells (PEC). Glands are surrounded by a thick layer of myoepithelial cells, stroma cells and collagen (arrows). H E staining × 200. 1B: High grade PIN in a p27-/- mice treated with MNU and sacrificed at the age of 9 months. Hyperplastic and dysplastic PEC cover entire glands and form papillary and alveolar structures (arrows). The surrounding connective tissue stroma is very abundant (arrow had). H E × 200. 1C: Ductal carcinoma of papillary type in DLP of a p27-/- animal treated with MNU and sacrificed at the age of 10 months. Note, tumor cells occupy the entire glands lumen. H E × 200. 1D: Infiltrative prostate carcinoma in a p27-/- animal sacrificed at the age of 12 months. Tumor cells infiltrate the surrounding stroma (arrows). H E × 200. 1E: Pituitary tumor in a p27-/- mice sacrificed at the age of 10 months. H E × 200. 1F: Intestinal carcinoma in a p27-/- mice sacrificed at the age of 9 months. Note tumor cell infiltration in the surrounding stroma (arrow). H E × 200. 1G: RARα expression in DLP of a 6-month-old p27+/- mice. RARα was detected by anti-RARα antibody (SC-251) and ABC kit. The receptor is expressed in papillary structures (arrow) and in the basal layer of epithelial cells. The slide is counterstained by hematoxylin × 200. 1H: RXRα expression in PIN of p27-/- 9-month-old mice. Note that most epithelial cells express RXRα receptor (arrow). The slide is counterstained by hematoxylin × 200.
Figure Legend Snippet: Morphological changes in DLP of p27 deficient mice in the course of carcinogenesis . 1A: Dorso-lateral prostate of 6-month-old p27-/- mice. Note the glandular structures are covered by a single layer of prostate epithelial cells (PEC). Glands are surrounded by a thick layer of myoepithelial cells, stroma cells and collagen (arrows). H E staining × 200. 1B: High grade PIN in a p27-/- mice treated with MNU and sacrificed at the age of 9 months. Hyperplastic and dysplastic PEC cover entire glands and form papillary and alveolar structures (arrows). The surrounding connective tissue stroma is very abundant (arrow had). H E × 200. 1C: Ductal carcinoma of papillary type in DLP of a p27-/- animal treated with MNU and sacrificed at the age of 10 months. Note, tumor cells occupy the entire glands lumen. H E × 200. 1D: Infiltrative prostate carcinoma in a p27-/- animal sacrificed at the age of 12 months. Tumor cells infiltrate the surrounding stroma (arrows). H E × 200. 1E: Pituitary tumor in a p27-/- mice sacrificed at the age of 10 months. H E × 200. 1F: Intestinal carcinoma in a p27-/- mice sacrificed at the age of 9 months. Note tumor cell infiltration in the surrounding stroma (arrow). H E × 200. 1G: RARα expression in DLP of a 6-month-old p27+/- mice. RARα was detected by anti-RARα antibody (SC-251) and ABC kit. The receptor is expressed in papillary structures (arrow) and in the basal layer of epithelial cells. The slide is counterstained by hematoxylin × 200. 1H: RXRα expression in PIN of p27-/- 9-month-old mice. Note that most epithelial cells express RXRα receptor (arrow). The slide is counterstained by hematoxylin × 200.

Techniques Used: Mouse Assay, Staining, Expressing

22) Product Images from "PML-RAR alpha induces the downmodulation of HHEX: a key event responsible for the induction of an angiogenetic response"

Article Title: PML-RAR alpha induces the downmodulation of HHEX: a key event responsible for the induction of an angiogenetic response

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-016-0262-5

Effect of ATO on downmodulation of HHEX expression elicited by PML-RARα induction in PR9 cells ( top and middle panels ) and on HHEX expression observed in NB4 cells ( bottom panel ). Top panels ( a , b ): PR9 cells have been grown either in the absence ( c ) or in the presence of ZnSO 4 for 48 h ( left and right panels ), in the presence of ATO for different times (from 6 to 48 h, right panel ), or in the presence of ZnSO 4 + ATO for different time points (from 6 to 48 h, left panel ) and HHEX, PML-RARα, or RARα expression were assessed by WB. A representative WB is shown at the top of each panel and the quantitative evaluation (mean ± SEM in three separate experiments). Bottom panel ( c ): NB4 cells have been grown either in the absence ( c ) or in the presence of ATO for different times (from 6 to 48 h) and HHEX expression was analyzed by WB
Figure Legend Snippet: Effect of ATO on downmodulation of HHEX expression elicited by PML-RARα induction in PR9 cells ( top and middle panels ) and on HHEX expression observed in NB4 cells ( bottom panel ). Top panels ( a , b ): PR9 cells have been grown either in the absence ( c ) or in the presence of ZnSO 4 for 48 h ( left and right panels ), in the presence of ATO for different times (from 6 to 48 h, right panel ), or in the presence of ZnSO 4 + ATO for different time points (from 6 to 48 h, left panel ) and HHEX, PML-RARα, or RARα expression were assessed by WB. A representative WB is shown at the top of each panel and the quantitative evaluation (mean ± SEM in three separate experiments). Bottom panel ( c ): NB4 cells have been grown either in the absence ( c ) or in the presence of ATO for different times (from 6 to 48 h) and HHEX expression was analyzed by WB

Techniques Used: Expressing, Western Blot

Effect of PML/RARα induction on the binding of PML-RARα to the HHEX gene promoter and on the expression of VEGF-A, VEGFR-2, and HHEX at RNA and at protein level. a PML-RARα binding to the HHEX promoter as shown by ChIP experiments. Nuclear extracts derived from PR9 cells grown for 24 h either in the absence ( c ) or in the presence of ZnSO 4 (Zn 2+ ); cells were crosslinked in vivo; after cell lysis, chromatin fragments were immunoprecipitated with anti-RARα antibody; and after DNA purification, DNA regions containing HHEX were amplified. In the figure, the HHEX PCR signal without immunoprecipitation is indicated as −A (IgG control) and, after immunoprecipitation, as +A (RARα antibody). b , c , d ZnSO 4 (Zn 2+ ) was added to PR9 cells and, at different time points and concentrations, cell aliquots were harvested and processed for evaluation of PML-RARα expression at protein level ( b ) and of VEGF-A, VEGFR-2, and HHEX expression at RNA level by RT-PCR ( c ) and at protein level by Western blotting ( d ). S26 was used for RT-PCR and β-actin for Western blotting normalization
Figure Legend Snippet: Effect of PML/RARα induction on the binding of PML-RARα to the HHEX gene promoter and on the expression of VEGF-A, VEGFR-2, and HHEX at RNA and at protein level. a PML-RARα binding to the HHEX promoter as shown by ChIP experiments. Nuclear extracts derived from PR9 cells grown for 24 h either in the absence ( c ) or in the presence of ZnSO 4 (Zn 2+ ); cells were crosslinked in vivo; after cell lysis, chromatin fragments were immunoprecipitated with anti-RARα antibody; and after DNA purification, DNA regions containing HHEX were amplified. In the figure, the HHEX PCR signal without immunoprecipitation is indicated as −A (IgG control) and, after immunoprecipitation, as +A (RARα antibody). b , c , d ZnSO 4 (Zn 2+ ) was added to PR9 cells and, at different time points and concentrations, cell aliquots were harvested and processed for evaluation of PML-RARα expression at protein level ( b ) and of VEGF-A, VEGFR-2, and HHEX expression at RNA level by RT-PCR ( c ) and at protein level by Western blotting ( d ). S26 was used for RT-PCR and β-actin for Western blotting normalization

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Derivative Assay, In Vivo, Lysis, Immunoprecipitation, DNA Purification, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

23) Product Images from "Targeting Expression of the Leukemogenic PML-RAR? Fusion Protein by Lentiviral Vector-Mediated Small Interfering RNA Results in Leukemic Cell Differentiation and Apoptosis"

Article Title: Targeting Expression of the Leukemogenic PML-RAR? Fusion Protein by Lentiviral Vector-Mediated Small Interfering RNA Results in Leukemic Cell Differentiation and Apoptosis

Journal: Human Gene Therapy

doi: 10.1089/hum.2011.079

Lentiviral vector-mediated delivery of siRNA targeting PML-RARα results in specific knockdown of expression. (A) The indicated lentiviral siRNA expression constructs were cotransfected into 293T cells with an expression construct for the long isoform of PML-RARα. siPML-RARα/S and siPML-RARα/L target the breakpoint regions of the short and long PML-RARα isoforms, respectively. si GFP, irrelevant control siRNA. Immunoblotting of whole cell extracts was performed with anti-PML antibody to detect PML-RARα ( right ). An anti-α-tubulin antibody was used to confirm equal protein loading ( bottom ). (B) The short isoform of PML-RARα was cotransfected with the indicated lentiviral siRNA expression constructs, followed by Western blotting of whole cell extracts using anti-PML and anti-α-tubulin antibodies. NS, nonspecific band. (C) Control 293T cells or 293T cells stably expressing the short isoform of PML-RARα were left untransduced (lanes 1 and 4), or were transduced with a lentiviral vector expressing siRNA specific for the short PML-RARα isoform (lanes 2 and 5) or with control lentiviral vector expressing only GFP (lanes 3 and 6). Whole cell extracts were prepared 5 days posttransduction and Western blotting was performed with anti-RARα antibody to detect PML-RARα and anti-α-tubulin antibody. (D) NB4 cells were mock transduced or transduced with the indicated lentiviral vectors delivering siRNA against the breakpoint regions of the short or long PML-RARα isoform. Whole cell extracts were prepared 5 days posttransduction and immunoblotting was performed with anti-PML, anti-RARα, and anti-α-tubulin antibodies.
Figure Legend Snippet: Lentiviral vector-mediated delivery of siRNA targeting PML-RARα results in specific knockdown of expression. (A) The indicated lentiviral siRNA expression constructs were cotransfected into 293T cells with an expression construct for the long isoform of PML-RARα. siPML-RARα/S and siPML-RARα/L target the breakpoint regions of the short and long PML-RARα isoforms, respectively. si GFP, irrelevant control siRNA. Immunoblotting of whole cell extracts was performed with anti-PML antibody to detect PML-RARα ( right ). An anti-α-tubulin antibody was used to confirm equal protein loading ( bottom ). (B) The short isoform of PML-RARα was cotransfected with the indicated lentiviral siRNA expression constructs, followed by Western blotting of whole cell extracts using anti-PML and anti-α-tubulin antibodies. NS, nonspecific band. (C) Control 293T cells or 293T cells stably expressing the short isoform of PML-RARα were left untransduced (lanes 1 and 4), or were transduced with a lentiviral vector expressing siRNA specific for the short PML-RARα isoform (lanes 2 and 5) or with control lentiviral vector expressing only GFP (lanes 3 and 6). Whole cell extracts were prepared 5 days posttransduction and Western blotting was performed with anti-RARα antibody to detect PML-RARα and anti-α-tubulin antibody. (D) NB4 cells were mock transduced or transduced with the indicated lentiviral vectors delivering siRNA against the breakpoint regions of the short or long PML-RARα isoform. Whole cell extracts were prepared 5 days posttransduction and immunoblotting was performed with anti-PML, anti-RARα, and anti-α-tubulin antibodies.

Techniques Used: Plasmid Preparation, Expressing, Construct, Western Blot, Stable Transfection, Transduction

Sequences of small interfering RNA (siRNA) specifically targeting the long and short isoforms of PML-RARα. Sequences at the breakpoint region are shown, with sequences contributed by PML and RARα indicated at the top . siRNA sequences are boxed. The breakpoint junction is indicated by the gap in the sequences; differences between siRNA sequences are underlined.
Figure Legend Snippet: Sequences of small interfering RNA (siRNA) specifically targeting the long and short isoforms of PML-RARα. Sequences at the breakpoint region are shown, with sequences contributed by PML and RARα indicated at the top . siRNA sequences are boxed. The breakpoint junction is indicated by the gap in the sequences; differences between siRNA sequences are underlined.

Techniques Used: Small Interfering RNA

24) Product Images from "High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis"

Article Title: High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis

Journal: Genes & Cancer

doi:

CRBP-1 transfection influences transcriptional pathways and differentiation of A549 adenocarcinoma cells A, bar graph after real-time PCR showing keratin (K) 1, 5, 14 and involucrin up-regulation, K10 down-regulation and unmodified K7 transcription level in CRBP-1 + compared to CRBP-1 − A549 cells. B, densitometric analysis of RARα, RARβ and RXRα protein expression by blot analysis. C, bar graph of RARs, RXRα, PPARβ/δ, FABP5 and CRABP-2 transcripts. D, bar graph and E, heat map of RT 2 profiler TM PCR assay of EGF/PDGF signaling-specific genes in CRBP-1 + A549 cells. Up-regulated and down-regulated genes are in dark grey and light grey, respectively. F, densitometric analysis of creb1, c-jun, p53, pAkt/Akt, pEGFR/EGFR and pErk1/2 protein expression by blots. Columns are means ± SEM of three different experiments. * p
Figure Legend Snippet: CRBP-1 transfection influences transcriptional pathways and differentiation of A549 adenocarcinoma cells A, bar graph after real-time PCR showing keratin (K) 1, 5, 14 and involucrin up-regulation, K10 down-regulation and unmodified K7 transcription level in CRBP-1 + compared to CRBP-1 − A549 cells. B, densitometric analysis of RARα, RARβ and RXRα protein expression by blot analysis. C, bar graph of RARs, RXRα, PPARβ/δ, FABP5 and CRABP-2 transcripts. D, bar graph and E, heat map of RT 2 profiler TM PCR assay of EGF/PDGF signaling-specific genes in CRBP-1 + A549 cells. Up-regulated and down-regulated genes are in dark grey and light grey, respectively. F, densitometric analysis of creb1, c-jun, p53, pAkt/Akt, pEGFR/EGFR and pErk1/2 protein expression by blots. Columns are means ± SEM of three different experiments. * p

Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

25) Product Images from "Ubiquitin-dependent Degradation of p73 Is Inhibited by PML"

Article Title: Ubiquitin-dependent Degradation of p73 Is Inhibited by PML

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20031943

p73 and PML colocalization within the PML-NBs is required for p73 stabilization (A) Representative image of Cos-1 cells costained with anti-p73 (green, clone 5B429) and anti-PML (red) antibodies and analyzed by confocal microscopy (left). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. The arrows indicate p73-containing speckles. (right) A quantitative analysis of the green and red fluorescence intensity at distinct nuclear speckles as indicated by the yellow arrow. (B–D) GFP-p73α was overexpressed into wild type (B) and Pml −/− (C) MEFs. Full-length PML was cotransfected with GFP-p73α into Pml −/− MEFs (D). Nuclei were visualized by DAPI staining. The arrows indicate p73-containing PML-NBs. (E) Pml −/− MEFs were transiently transfected with HA-p73α alone (lane 1) or in combination with full-length Flag-PML (lane 2), Flag-PML ΔRING (lane 3), or PML-RARα (lane 4). Cell extracts were subjected to IB with anti-HA, anti-Flag, anti-RARα, and anti–β-actin antibodies. (bottom) Normalization of transfection efficiency by quantitation of GFP expression.
Figure Legend Snippet: p73 and PML colocalization within the PML-NBs is required for p73 stabilization (A) Representative image of Cos-1 cells costained with anti-p73 (green, clone 5B429) and anti-PML (red) antibodies and analyzed by confocal microscopy (left). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. The arrows indicate p73-containing speckles. (right) A quantitative analysis of the green and red fluorescence intensity at distinct nuclear speckles as indicated by the yellow arrow. (B–D) GFP-p73α was overexpressed into wild type (B) and Pml −/− (C) MEFs. Full-length PML was cotransfected with GFP-p73α into Pml −/− MEFs (D). Nuclei were visualized by DAPI staining. The arrows indicate p73-containing PML-NBs. (E) Pml −/− MEFs were transiently transfected with HA-p73α alone (lane 1) or in combination with full-length Flag-PML (lane 2), Flag-PML ΔRING (lane 3), or PML-RARα (lane 4). Cell extracts were subjected to IB with anti-HA, anti-Flag, anti-RARα, and anti–β-actin antibodies. (bottom) Normalization of transfection efficiency by quantitation of GFP expression.

Techniques Used: Confocal Microscopy, Staining, Fluorescence, Transfection, Quantitation Assay, Expressing

PML-regulated acetylation of p73 by p300 protects p73 against degradation. (A) GFP-p73α and HA-Ub were expressed in 293T cells in the absence (lane 3) or in the presence (lane 4) of p300bromo-HAT for 24 h. Cells were incubated with 1 μM trichostatin A (TSA) and 1 mM niacinamide for the last 12 h of transfection. After IP of GFP-p73, ubiquitinylation levels of p73 were detected with anti-HA antibody. Acetylated and unacetylated p73 in the immunoprecipitates were determined with anti–acetyl-lysine and anti-GFP antibody, respectively. (B) 293T cells were transfected with a plasmid encoding GFP-p73α alone (lane 1) or in combination with p300bromo-HAT (lanes 2–6), in the absence (lane 2) or in the presence (lanes 3–6) of decreasing doses of HA-Ub for 24 h. Treatment with deacetylase inhibitors and IP experiments were performed as described in A. (C and D) Transformed 3T3 Pml −/− MEFs were transiently transfected with GFP-p73α alone (lane 1) or along with p300 in the absence (lane 2) or in the presence of full-length Flag-PML (lane 3), Flag-PML-ΔRING (lane 4), or Flag-PML-RARα (lane 5) for 24 h. Cells were incubated with deacetylase inhibitors for 8 h. p73 was immunoprecipitated using anti-GFP antibody followed by IB with anti–acetyl-lysine antibody (C). Whole cell extracts were immunoblotted with anti-GFP, anti-Flag, and anti–β-actin antibodies (D).
Figure Legend Snippet: PML-regulated acetylation of p73 by p300 protects p73 against degradation. (A) GFP-p73α and HA-Ub were expressed in 293T cells in the absence (lane 3) or in the presence (lane 4) of p300bromo-HAT for 24 h. Cells were incubated with 1 μM trichostatin A (TSA) and 1 mM niacinamide for the last 12 h of transfection. After IP of GFP-p73, ubiquitinylation levels of p73 were detected with anti-HA antibody. Acetylated and unacetylated p73 in the immunoprecipitates were determined with anti–acetyl-lysine and anti-GFP antibody, respectively. (B) 293T cells were transfected with a plasmid encoding GFP-p73α alone (lane 1) or in combination with p300bromo-HAT (lanes 2–6), in the absence (lane 2) or in the presence (lanes 3–6) of decreasing doses of HA-Ub for 24 h. Treatment with deacetylase inhibitors and IP experiments were performed as described in A. (C and D) Transformed 3T3 Pml −/− MEFs were transiently transfected with GFP-p73α alone (lane 1) or along with p300 in the absence (lane 2) or in the presence of full-length Flag-PML (lane 3), Flag-PML-ΔRING (lane 4), or Flag-PML-RARα (lane 5) for 24 h. Cells were incubated with deacetylase inhibitors for 8 h. p73 was immunoprecipitated using anti-GFP antibody followed by IB with anti–acetyl-lysine antibody (C). Whole cell extracts were immunoblotted with anti-GFP, anti-Flag, and anti–β-actin antibodies (D).

Techniques Used: HAT Assay, Incubation, Transfection, Plasmid Preparation, Histone Deacetylase Assay, Transformation Assay, Immunoprecipitation

26) Product Images from "Familial Focal Segmental Glomerulosclerosis (FSGS)-linked ?-Actinin 4 (ACTN4) Protein Mutants Lose Ability to Activate Transcription by Nuclear Hormone Receptors *"

Article Title: Familial Focal Segmental Glomerulosclerosis (FSGS)-linked ?-Actinin 4 (ACTN4) Protein Mutants Lose Ability to Activate Transcription by Nuclear Hormone Receptors *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.345421

ACTN4 binds and potentiates transcriptional activation by RARα. A , Western blot of co-immunoprecipitated samples demonstrating an interaction between ACTN4 (WT) and RARα. HEK293 cells were transfected with HA-RARα expression plasmids.
Figure Legend Snippet: ACTN4 binds and potentiates transcriptional activation by RARα. A , Western blot of co-immunoprecipitated samples demonstrating an interaction between ACTN4 (WT) and RARα. HEK293 cells were transfected with HA-RARα expression plasmids.

Techniques Used: Activation Assay, Western Blot, Immunoprecipitation, Transfection, Expressing

FSGS-linked ACTN4 mutants lose their interactions with nuclear hormone receptors. A , FSGS-linked ACTN4 mutant K228E fails to interact with RARα in HEK293 cells. FLAG-ACTN4 was co-transfected with or without HA-RARα into HEK293 cells. Whole
Figure Legend Snippet: FSGS-linked ACTN4 mutants lose their interactions with nuclear hormone receptors. A , FSGS-linked ACTN4 mutant K228E fails to interact with RARα in HEK293 cells. FLAG-ACTN4 was co-transfected with or without HA-RARα into HEK293 cells. Whole

Techniques Used: Mutagenesis, Transfection

FSGS-linked ACTN4 mutants lose their ability to potentiate RARα-mediated transcription activation. A , schematic diagram of ACTN4 indicating the position of FSGS-linked mutations. ACTN4 harbors two CH domains, CH1 and CH2, four spectrin repeats
Figure Legend Snippet: FSGS-linked ACTN4 mutants lose their ability to potentiate RARα-mediated transcription activation. A , schematic diagram of ACTN4 indicating the position of FSGS-linked mutations. ACTN4 harbors two CH domains, CH1 and CH2, four spectrin repeats

Techniques Used: Activation Assay

27) Product Images from "Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription *"

Article Title: Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.132795

Schematic presentation of proposed interactions between regulatory elements during ATRA-mediated regulation of the Npr1 gene. The proposed diagram indicates that ATRA increases Npr1 gene transcription and expression in association of Ets-1, Sp1, histone acetylation. ATRA induces Ets-1 protein expression via its receptor RARα and enhances Ets-1 and Sp1 binding to the Npr1 promoter. In the absence of functional RARE, RARα associates with the Ets-1·Sp1 complex on the Npr1 promoter and mediates ATRA effects. Furthermore, ATRA acetylates histone H3 and H4 around the Ets-1 and Sp1 binding sites in the Npr1 promoter. Histone acetylation promotes localized unwinding of DNA and allows transcription factors to bind in the region. RA , retinoic acid. The closed upward arrow indicates an increased Ets-1 protein expression; the open upward arrow indicates an increased binding of Ets-1·Sp1·RARα complex to the Npr1 gene promoter.
Figure Legend Snippet: Schematic presentation of proposed interactions between regulatory elements during ATRA-mediated regulation of the Npr1 gene. The proposed diagram indicates that ATRA increases Npr1 gene transcription and expression in association of Ets-1, Sp1, histone acetylation. ATRA induces Ets-1 protein expression via its receptor RARα and enhances Ets-1 and Sp1 binding to the Npr1 promoter. In the absence of functional RARE, RARα associates with the Ets-1·Sp1 complex on the Npr1 promoter and mediates ATRA effects. Furthermore, ATRA acetylates histone H3 and H4 around the Ets-1 and Sp1 binding sites in the Npr1 promoter. Histone acetylation promotes localized unwinding of DNA and allows transcription factors to bind in the region. RA , retinoic acid. The closed upward arrow indicates an increased Ets-1 protein expression; the open upward arrow indicates an increased binding of Ets-1·Sp1·RARα complex to the Npr1 gene promoter.

Techniques Used: Expressing, Binding Assay, Functional Assay

28) Product Images from "Induction of miR-21 by Retinoic Acid in Estrogen Receptor-positive Breast Carcinoma Cells"

Article Title: Induction of miR-21 by Retinoic Acid in Estrogen Receptor-positive Breast Carcinoma Cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.184994

Transcriptional regulation of MIR21 by retinoids in ERα + cell lines via RARα. A , MCF-7 cells were treated with ATRA (1 μ m ) or vehicle (Me 2 SO) for 48 h. Upper panel , RT-PCR analysis of pri-miR-21, using a primer pair corresponding
Figure Legend Snippet: Transcriptional regulation of MIR21 by retinoids in ERα + cell lines via RARα. A , MCF-7 cells were treated with ATRA (1 μ m ) or vehicle (Me 2 SO) for 48 h. Upper panel , RT-PCR analysis of pri-miR-21, using a primer pair corresponding

Techniques Used: Reverse Transcription Polymerase Chain Reaction

Significance of RARα for miR-21 induction by ATRA and retinoid sensitivity in MCF-7. A , the panel shows the levels of the transcripts encoding RAR and RXR nuclear retinoic acid receptors in MCF-7 and MDA-MB-231 cells. The results were obtained
Figure Legend Snippet: Significance of RARα for miR-21 induction by ATRA and retinoid sensitivity in MCF-7. A , the panel shows the levels of the transcripts encoding RAR and RXR nuclear retinoic acid receptors in MCF-7 and MDA-MB-231 cells. The results were obtained

Techniques Used: Multiple Displacement Amplification

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Blocking Assay:

Article Title: Regulation of cyp26a1 on Th17 cells in mouse peri-implantation
Article Snippet: After blocking in horse serum for 1 h at 37°C, the sections were incubated with first antibody that diluted in PBS overnight at 4°C. .. Here is the primary and secondary antibodies list used in immunohistochemistry: anti-RORγt (17-6988, eBioscience, USA), anti-IL-17 (sc-7929, Santa Cruz,USA), anti-cyp26a1 (CYP26A12-A, Acris, Hiddenhausen, Germany), anti-RARα (sc-15040, Santa Cruz, USA); goat anti-rat (112-035-003, Jackson ImmunoResaerch, USA), goat anti-rabbit (074-1506, KPL, USA), rabbit anti-goat (14-13-06, KPL, USA).

SYBR Green Assay:

Article Title: BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid
Article Snippet: DNA protein complexes were immunoprecipitated using 4 µg anti-BATF (rabbit polyclonal; Cell Signaling Technology), anti-RARα (rabbit polyclonal; Santa Cruz Biotechnology, Inc.), and anti–acetyl-histone H4 antibodies (EMD Millipore). .. Real-time PCR detection using the primers described in Table S3 was conducted with a 7500 Sequence Detection System using the SYBR green Master Mix (Applied Biosystems).

Incubation:

Article Title: Kr?ppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor ?-mediated, Not by Retinoic Acid Receptor ?-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells *
Article Snippet: .. The membranes were blocked with 5% milk in TTBS for 2 h at room temperature and incubated overnight at 4 °C using the following primary antibodies: 1:500 rabbit anti-Klf4 (Santa Cruz), 1:500 mouse anti-ERK1/2 (Santa Cruz), 1:500 mouse antiphospho-ERK1/2 (Santa Cruz), 1:1000 rabbit anti-MEK1/2 (Cell Signaling), 1:1000 rabbit antiphospho-MEK1/2 (Cell Signaling), 1:1000 rabbit anti-p38 (Cell Signaling), 1:500 rabbit anti-PDGFR (Santa Cruz), 1:200 antiphospho-PDGFRβ (Santa Cruz), 1:500 anti-RARα (Santa Cruz), 1:500 anti-retinoic X receptor (RXRα) (Santa Cruz), and 1:1000 mouse anti-β-actin (Santa Cruz). .. The membranes were then incubated for 1 h at room temperature with a 1:5000 dilution of anti-rabbit/horseradish peroxidase or anti-mouse/horseradish peroxidase (Santa Cruz) and developed with the Chemiluminescence Plus Western blot analysis kit (Santa Cruz).

Article Title: Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system
Article Snippet: Anti-chromogranin A (Abcam ab15160, 1:3000), anti-RARα (Santa Cruz biotechnologies, 1:500), anti-Cre-recombinase (Abcam, 1:125), anti-cleaved caspase 3 (Cell signaling technologies, 1:400), anti-OLFM4 (Cell signaling technologies, 1:400), anti KLF4 (R & D Systems, 1:200) and anti-Ki67 (Biolegend, 16A8, 1:50) antibodies were used for immunohistochemistry. .. Borg decloaker solution was used for antigen retrieval prior anti-Lysozyme antibody incubation (Thermo Ab-1 RB-372-A1, 1:2000) for Paneth cells detection.

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Article Snippet: Streptavidin-Sepharose beads were then added and incubated with these mixtures for an additional 30 min at 4°C. .. Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs.

Article Title: Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription
Article Snippet: For the super shift reaction 1 µg of anti-RARα (C-20, Santa Cruz) or anti-RXR (F-1, Santa Cruz) antibodies were included to the reaction just before adding the Biotin labeled oligos ( Supplementary Table S2 ). .. Binding reactions were incubated at room temperature for 40 min and then resolved on a 6% native polyacrylamide gel in 0.5× TBE in a minigel electrophoresis apparatus (dimensions 8 × 8 × 0.1 cm).

Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression
Article Snippet: For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA). .. Briefly, 400 μg of protein in 100 μl of lysis buffer was incubated with the selected antibodies overnight at 4 °C with rotation.

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: .. The blotted membranes were then incubated with antibodies against PLZF, RARα (Santa Cruz), GAPDH (Chemicon), p21 (Cell Signaling), p53, HDAC1, NCoR, HDAC3, PLZF, and SMRT (Santa Cruz) and further incubated with either an anti-mouse or anti-rabbit secondary antibody conjugated to HRP (Vector Laboratories). .. Finally, antibody-bound protein bands were visualized using an ECL solution (PerkinElmer Life Sciences).

Article Title: Regulation of cyp26a1 on Th17 cells in mouse peri-implantation
Article Snippet: After washing thoroughly in PBS, the sections were incubated with a secondary antibody (112-035-003, Jackson ImmunoResearch, USA) diluted 1:400 in PBS at 37°C for 2 h. The colour was developed using diaminobenzidine tetrahydrochloride (Sigma–Aldrich, USA). .. Here is the primary and secondary antibodies list used in immunohistochemistry: anti-RORγt (17-6988, eBioscience, USA), anti-IL-17 (sc-7929, Santa Cruz,USA), anti-cyp26a1 (CYP26A12-A, Acris, Hiddenhausen, Germany), anti-RARα (sc-15040, Santa Cruz, USA); goat anti-rat (112-035-003, Jackson ImmunoResaerch, USA), goat anti-rabbit (074-1506, KPL, USA), rabbit anti-goat (14-13-06, KPL, USA).

Activity Assay:

Article Title: Regulation of cyp26a1 on Th17 cells in mouse peri-implantation
Article Snippet: Immunohistochemistry The frozen sections (8 μm) of mouse uterus implantation sits were mounted on 3-aminopropyltriethoxy-silane–coated slides and fixed in 4% PFA for 10 min. After washing the sections with PBS, endogenous peroxidase activity was blocked by 0.3% H2 O2 for 10 min at room temperature. .. Here is the primary and secondary antibodies list used in immunohistochemistry: anti-RORγt (17-6988, eBioscience, USA), anti-IL-17 (sc-7929, Santa Cruz,USA), anti-cyp26a1 (CYP26A12-A, Acris, Hiddenhausen, Germany), anti-RARα (sc-15040, Santa Cruz, USA); goat anti-rat (112-035-003, Jackson ImmunoResaerch, USA), goat anti-rabbit (074-1506, KPL, USA), rabbit anti-goat (14-13-06, KPL, USA).

Expressing:

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Article Snippet: COS-7 cells were transfected with 1 µg of expression vectors using LipofectAmine 2000 (Invitrogen), according to the manufacturer's instructions. .. Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs.

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: HEK293, HCT116 p53+/+ , HCT116 p53−/− , and HL-60 cells were transfected with increasing amounts of pSG5-PLZF-RARα expression vector. .. The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

Western Blot:

Article Title: Kr?ppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor ?-mediated, Not by Retinoic Acid Receptor ?-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells *
Article Snippet: Paragraph title: Western Blot Analysis ... The membranes were blocked with 5% milk in TTBS for 2 h at room temperature and incubated overnight at 4 °C using the following primary antibodies: 1:500 rabbit anti-Klf4 (Santa Cruz), 1:500 mouse anti-ERK1/2 (Santa Cruz), 1:500 mouse antiphospho-ERK1/2 (Santa Cruz), 1:1000 rabbit anti-MEK1/2 (Cell Signaling), 1:1000 rabbit antiphospho-MEK1/2 (Cell Signaling), 1:1000 rabbit anti-p38 (Cell Signaling), 1:500 rabbit anti-PDGFR (Santa Cruz), 1:200 antiphospho-PDGFRβ (Santa Cruz), 1:500 anti-RARα (Santa Cruz), 1:500 anti-retinoic X receptor (RXRα) (Santa Cruz), and 1:1000 mouse anti-β-actin (Santa Cruz).

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Article Snippet: .. Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs. .. Transfection and luciferase assay COS-7 cells were maintained in complete DMEM medium.

Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression
Article Snippet: Paragraph title: Western Blot Immunoprecipitation and siRNA ... For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA).

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: Paragraph title: Western Blot Analysis ... The blotted membranes were then incubated with antibodies against PLZF, RARα (Santa Cruz), GAPDH (Chemicon), p21 (Cell Signaling), p53, HDAC1, NCoR, HDAC3, PLZF, and SMRT (Santa Cruz) and further incubated with either an anti-mouse or anti-rabbit secondary antibody conjugated to HRP (Vector Laboratories).

Article Title: Targeting the Acute Promyelocytic Leukemia-Associated Fusion Proteins PML/RAR? and PLZF/RAR? with Interfering Peptides
Article Snippet: .. Western blot analysis was performed using the anti-RARα (C-20 - Santa Cruz Biotechnology, Santa Cruz, USA), anti-GFP (FL - St. Cruz), anti-c-ABL (24-11, St. Cruz Biotechnology, Santa Cruz, USA) and anti-phospho-ABL (Tyr245, Millipore, Billerica, USA), anti-GAPDH (FL-335 - St. Cruz), and anti-tubulin (Ab-4, NeoMarkers, Thermo Scientific Inc., Kalamazoo, USA) antibodies. ..

Article Title: The Ski protein can inhibit ligand induced RAR? and HDAC3 degradation in the Retinoic acid signaling pathway
Article Snippet: .. Co-immunoprecipitation and western blot were performed as described [ ] Antibodies used were anti-Myc (9E10, Santa Cruz Biotechnology, CA, USA), anti-Flag mouse monoclonal antibody (Sigma), anti-T7 mouse monoclonal antibodies (Novagen, Madison, WI, USA), anti-Ski (H-329, Santa Cruz Biotechnology), anti-HDAC3 (3G6, Upstate, Charlottesville, VA, USA), anti-Ski (G8, Cascade Bioscience, Winchester, MA, USA), anti-RARα (Santa Cruz Biotechnology), anti-β-Actin (Sigma), and anti-α-Tubulin (Sigma). .. Proteins were detected by chemiluminescence (Perkin Elmer, Shelton, CT, USA).

Real-time Polymerase Chain Reaction:

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz). .. Quantitative PCR of the chromatin-immunoprecipitated DNA was performed using the following oligonucleotide primer sets, which were designed to amplify the upstream regulatory regions flanking the p53 and RARα binding sites and the CDKN1A proximal promoter region: p53RE-1 (bp −2307 to −1947) forward, 5′-CTGTGGCTCTGATTGGCTTT-3′ and reverse, 5′-GGGTCTTTAGAGGTCTCCTGTCT-3′; p53RE-2 (bp −1462 to −1128) forward, 5′-CCACAGCAGAGGAGAAAGAAG-3′ and reverse, 5′-GCTGCTCAGAGTCTGGAAATC-3′; RARE (bp −1020 to −1200) forward, 5′-GGGTTCTGTTTTTTAGTGGGATTTC-3′ and reverse, 5′-TGGAAGTGTCTACTGGTTCTTCTGA-3′; and CDKN1A proximal promoter (bp −131 to +30) forward, 5′-GCGCTGGGCAGCCAGGAGCCT-3′ and reverse, 5′-CTGACTTCGGCAGCTGCTCAC-3′.

Article Title: BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid
Article Snippet: DNA protein complexes were immunoprecipitated using 4 µg anti-BATF (rabbit polyclonal; Cell Signaling Technology), anti-RARα (rabbit polyclonal; Santa Cruz Biotechnology, Inc.), and anti–acetyl-histone H4 antibodies (EMD Millipore). .. Real-time PCR detection using the primers described in Table S3 was conducted with a 7500 Sequence Detection System using the SYBR green Master Mix (Applied Biosystems).

Immunohistochemistry:

Article Title: Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system
Article Snippet: .. Anti-chromogranin A (Abcam ab15160, 1:3000), anti-RARα (Santa Cruz biotechnologies, 1:500), anti-Cre-recombinase (Abcam, 1:125), anti-cleaved caspase 3 (Cell signaling technologies, 1:400), anti-OLFM4 (Cell signaling technologies, 1:400), anti KLF4 (R & D Systems, 1:200) and anti-Ki67 (Biolegend, 16A8, 1:50) antibodies were used for immunohistochemistry. .. Borg decloaker solution was used for antigen retrieval prior anti-Lysozyme antibody incubation (Thermo Ab-1 RB-372-A1, 1:2000) for Paneth cells detection.

Article Title: Regulation of cyp26a1 on Th17 cells in mouse peri-implantation
Article Snippet: .. Here is the primary and secondary antibodies list used in immunohistochemistry: anti-RORγt (17-6988, eBioscience, USA), anti-IL-17 (sc-7929, Santa Cruz,USA), anti-cyp26a1 (CYP26A12-A, Acris, Hiddenhausen, Germany), anti-RARα (sc-15040, Santa Cruz, USA); goat anti-rat (112-035-003, Jackson ImmunoResaerch, USA), goat anti-rabbit (074-1506, KPL, USA), rabbit anti-goat (14-13-06, KPL, USA). .. Isolation of Mononuclear Cells from spleen and peripheral blood Blood was drawn from the eyeballs and diluted with a volume of onefold Hank's solution for mononuclear cell isolation.

Immunoprecipitation:

Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression
Article Snippet: Paragraph title: Western Blot Immunoprecipitation and siRNA ... For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA).

Article Title: BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid
Article Snippet: .. DNA protein complexes were immunoprecipitated using 4 µg anti-BATF (rabbit polyclonal; Cell Signaling Technology), anti-RARα (rabbit polyclonal; Santa Cruz Biotechnology, Inc.), and anti–acetyl-histone H4 antibodies (EMD Millipore). .. Real-time PCR detection using the primers described in Table S3 was conducted with a 7500 Sequence Detection System using the SYBR green Master Mix (Applied Biosystems).

Protease Inhibitor:

Article Title: Kr?ppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor ?-mediated, Not by Retinoic Acid Receptor ?-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells *
Article Snippet: The cells were lysed with 150 m m NaCl, 50 m m Tris-HCl (pH 7.5), 1% Nonidet P-40, 0.5% sodium deoxycholic acid, and Complete protease inhibitor mixture tablets (Roche Applied Science), and the protein was then isolated ( , ). .. The membranes were blocked with 5% milk in TTBS for 2 h at room temperature and incubated overnight at 4 °C using the following primary antibodies: 1:500 rabbit anti-Klf4 (Santa Cruz), 1:500 mouse anti-ERK1/2 (Santa Cruz), 1:500 mouse antiphospho-ERK1/2 (Santa Cruz), 1:1000 rabbit anti-MEK1/2 (Cell Signaling), 1:1000 rabbit antiphospho-MEK1/2 (Cell Signaling), 1:1000 rabbit anti-p38 (Cell Signaling), 1:500 rabbit anti-PDGFR (Santa Cruz), 1:200 antiphospho-PDGFRβ (Santa Cruz), 1:500 anti-RARα (Santa Cruz), 1:500 anti-retinoic X receptor (RXRα) (Santa Cruz), and 1:1000 mouse anti-β-actin (Santa Cruz).

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Article Snippet: Transfected COS-7 cells or nuclear fractions of BM-DCs were lysed or diluted with DNAP binding buffer [25 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.25% NP-40, 1 mM DTT and Complete Protease Inhibitor Cocktail (Nacalai Tesque)], respectively. .. Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs.

Cell Culture:

Article Title: BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid
Article Snippet: CD4+ T cells (5 × 106 naive per sample), cultured in the presence of 100 U/ml IL-2, 1.5 µg/ml concanavalin A, and 10 nM RA for 4–5 d, were used for ChIP assays. .. DNA protein complexes were immunoprecipitated using 4 µg anti-BATF (rabbit polyclonal; Cell Signaling Technology), anti-RARα (rabbit polyclonal; Santa Cruz Biotechnology, Inc.), and anti–acetyl-histone H4 antibodies (EMD Millipore).

Article Title: Targeting the Acute Promyelocytic Leukemia-Associated Fusion Proteins PML/RAR? and PLZF/RAR? with Interfering Peptides
Article Snippet: Paragraph title: Cell lines, cell culture and western blotting ... Western blot analysis was performed using the anti-RARα (C-20 - Santa Cruz Biotechnology, Santa Cruz, USA), anti-GFP (FL - St. Cruz), anti-c-ABL (24-11, St. Cruz Biotechnology, Santa Cruz, USA) and anti-phospho-ABL (Tyr245, Millipore, Billerica, USA), anti-GAPDH (FL-335 - St. Cruz), and anti-tubulin (Ab-4, NeoMarkers, Thermo Scientific Inc., Kalamazoo, USA) antibodies.

Oligonucleotide Labeling:

Article Title: Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription
Article Snippet: Oligonucleotide labeling was performed using the Biotin 3′ End Labeling Kit (Pierce Biotechnology). .. For the super shift reaction 1 µg of anti-RARα (C-20, Santa Cruz) or anti-RXR (F-1, Santa Cruz) antibodies were included to the reaction just before adding the Biotin labeled oligos ( Supplementary Table S2 ).

Sequencing:

Article Title: BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid
Article Snippet: DNA protein complexes were immunoprecipitated using 4 µg anti-BATF (rabbit polyclonal; Cell Signaling Technology), anti-RARα (rabbit polyclonal; Santa Cruz Biotechnology, Inc.), and anti–acetyl-histone H4 antibodies (EMD Millipore). .. Real-time PCR detection using the primers described in Table S3 was conducted with a 7500 Sequence Detection System using the SYBR green Master Mix (Applied Biosystems).

Binding Assay:

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Article Snippet: After washing the beads three times in DNAP binding buffer, precipitated proteins were eluted in SDS-PAGE sample buffer. .. Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs.

Article Title: Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription
Article Snippet: In brief, binding reactions included 1 × binding buffer, 2.5% glycerol, 5 mM MgCl2 , 50 ng/µl Poly (dI•dC), 0.05% NP-40, 4 pmol of unlabeled oligo, 5 µl nuclear extract and 20 fmol of biotin labeled oligo. .. For the super shift reaction 1 µg of anti-RARα (C-20, Santa Cruz) or anti-RXR (F-1, Santa Cruz) antibodies were included to the reaction just before adding the Biotin labeled oligos ( Supplementary Table S2 ).

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: .. The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz). .. The level of endogenous Sp1 and p53 protein binding was analyzed using polyclonal antibodies against anti-p53 (Santa Cruz), anti-Sp1 (Santa Cruz), anti-Ac-Histone 3, anti-Ac-Histone 4, anti-Histone H3K4-Me3, and anti-Histone H3K9-Me3 (Upstate).

Affinity Precipitation:

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Article Snippet: Paragraph title: DNA affinity precipitation (DNAP) assay ... Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs.

Isolation:

Article Title: Kr?ppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor ?-mediated, Not by Retinoic Acid Receptor ?-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells *
Article Snippet: The cells were lysed with 150 m m NaCl, 50 m m Tris-HCl (pH 7.5), 1% Nonidet P-40, 0.5% sodium deoxycholic acid, and Complete protease inhibitor mixture tablets (Roche Applied Science), and the protein was then isolated ( , ). .. The membranes were blocked with 5% milk in TTBS for 2 h at room temperature and incubated overnight at 4 °C using the following primary antibodies: 1:500 rabbit anti-Klf4 (Santa Cruz), 1:500 mouse anti-ERK1/2 (Santa Cruz), 1:500 mouse antiphospho-ERK1/2 (Santa Cruz), 1:1000 rabbit anti-MEK1/2 (Cell Signaling), 1:1000 rabbit antiphospho-MEK1/2 (Cell Signaling), 1:1000 rabbit anti-p38 (Cell Signaling), 1:500 rabbit anti-PDGFR (Santa Cruz), 1:200 antiphospho-PDGFRβ (Santa Cruz), 1:500 anti-RARα (Santa Cruz), 1:500 anti-retinoic X receptor (RXRα) (Santa Cruz), and 1:1000 mouse anti-β-actin (Santa Cruz).

Transfection:

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Article Snippet: Transfected COS-7 cells or nuclear fractions of BM-DCs were lysed or diluted with DNAP binding buffer [25 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.25% NP-40, 1 mM DTT and Complete Protease Inhibitor Cocktail (Nacalai Tesque)], respectively. .. Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs.

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: HEK293, HCT116 p53+/+ , HCT116 p53−/− , and HL-60 cells were transfected with increasing amounts of pSG5-PLZF-RARα expression vector. .. The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

Labeling:

Article Title: Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription
Article Snippet: .. For the super shift reaction 1 µg of anti-RARα (C-20, Santa Cruz) or anti-RXR (F-1, Santa Cruz) antibodies were included to the reaction just before adding the Biotin labeled oligos ( Supplementary Table S2 ). .. Binding reactions were incubated at room temperature for 40 min and then resolved on a 6% native polyacrylamide gel in 0.5× TBE in a minigel electrophoresis apparatus (dimensions 8 × 8 × 0.1 cm).

Electrophoretic Mobility Shift Assay:

Article Title: Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription
Article Snippet: Paragraph title: Electrophoretic mobility shift assay ... For the super shift reaction 1 µg of anti-RARα (C-20, Santa Cruz) or anti-RXR (F-1, Santa Cruz) antibodies were included to the reaction just before adding the Biotin labeled oligos ( Supplementary Table S2 ).

Staining:

Article Title: Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system
Article Snippet: Anti-chromogranin A (Abcam ab15160, 1:3000), anti-RARα (Santa Cruz biotechnologies, 1:500), anti-Cre-recombinase (Abcam, 1:125), anti-cleaved caspase 3 (Cell signaling technologies, 1:400), anti-OLFM4 (Cell signaling technologies, 1:400), anti KLF4 (R & D Systems, 1:200) and anti-Ki67 (Biolegend, 16A8, 1:50) antibodies were used for immunohistochemistry. .. For goblet cell staining, slides were incubated in 1% Alcian Blue/3% acetic acid solution for five minutes and counterstained with nuclear fast red.

Chromatin Immunoprecipitation:

Article Title: PU.1 controls the expression of long noncoding RNA HOTAIRM1 during granulocytic differentiation
Article Snippet: Paragraph title: Chromatin immunoprecipitation assay ... The following antibodies were used: anti-PU.1 (#2258; Cell Signaling Technology, Danvers, MA, USA), anti-RARα (C-20 X; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-PML (H238 X; Santa Cruz).

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: .. The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz). .. The level of endogenous Sp1 and p53 protein binding was analyzed using polyclonal antibodies against anti-p53 (Santa Cruz), anti-Sp1 (Santa Cruz), anti-Ac-Histone 3, anti-Ac-Histone 4, anti-Histone H3K4-Me3, and anti-Histone H3K9-Me3 (Upstate).

Article Title: BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid
Article Snippet: Paragraph title: Bioinformatics and ChIP assays. ... DNA protein complexes were immunoprecipitated using 4 µg anti-BATF (rabbit polyclonal; Cell Signaling Technology), anti-RARα (rabbit polyclonal; Santa Cruz Biotechnology, Inc.), and anti–acetyl-histone H4 antibodies (EMD Millipore).

Article Title: Thymosin‐β4 is a determinant of drug sensitivity for Fenretinide and Vorinostat combination therapy in neuroblastoma), Thymosin-β4 is a determinant of drug sensitivity for Fenretinide and Vorinostat combination therapy in neuroblastoma
Article Snippet: Paragraph title: 2.10. Chromatin immuno‐precipitation (ChIP) assays ... Antibodies used in this study were as follows: IgG (sc‐2027, Santa Cruz Biotechnology, Santa Cruz, CA), RARα (Santa Cruz Biotechnology).

SDS Page:

Article Title: Kr?ppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor ?-mediated, Not by Retinoic Acid Receptor ?-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells *
Article Snippet: Total protein (70 μg) from each sample was separated by 8% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore). .. The membranes were blocked with 5% milk in TTBS for 2 h at room temperature and incubated overnight at 4 °C using the following primary antibodies: 1:500 rabbit anti-Klf4 (Santa Cruz), 1:500 mouse anti-ERK1/2 (Santa Cruz), 1:500 mouse antiphospho-ERK1/2 (Santa Cruz), 1:1000 rabbit anti-MEK1/2 (Cell Signaling), 1:1000 rabbit antiphospho-MEK1/2 (Cell Signaling), 1:1000 rabbit anti-p38 (Cell Signaling), 1:500 rabbit anti-PDGFR (Santa Cruz), 1:200 antiphospho-PDGFRβ (Santa Cruz), 1:500 anti-RARα (Santa Cruz), 1:500 anti-retinoic X receptor (RXRα) (Santa Cruz), and 1:1000 mouse anti-β-actin (Santa Cruz).

Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Article Snippet: .. Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs. .. Transfection and luciferase assay COS-7 cells were maintained in complete DMEM medium.

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: The cell extracts (40 μg) were separated using a 12% SDS-PAGE gel, transferred onto Immuno-BlotTM PVDF membranes (Bio-Rad), and blocked with 5% skim milk (BD Biosciences). .. The blotted membranes were then incubated with antibodies against PLZF, RARα (Santa Cruz), GAPDH (Chemicon), p21 (Cell Signaling), p53, HDAC1, NCoR, HDAC3, PLZF, and SMRT (Santa Cruz) and further incubated with either an anti-mouse or anti-rabbit secondary antibody conjugated to HRP (Vector Laboratories).

Plasmid Preparation:

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: HEK293, HCT116 p53+/+ , HCT116 p53−/− , and HL-60 cells were transfected with increasing amounts of pSG5-PLZF-RARα expression vector. .. The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

Software:

Article Title: Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system
Article Snippet: Anti-chromogranin A (Abcam ab15160, 1:3000), anti-RARα (Santa Cruz biotechnologies, 1:500), anti-Cre-recombinase (Abcam, 1:125), anti-cleaved caspase 3 (Cell signaling technologies, 1:400), anti-OLFM4 (Cell signaling technologies, 1:400), anti KLF4 (R & D Systems, 1:200) and anti-Ki67 (Biolegend, 16A8, 1:50) antibodies were used for immunohistochemistry. .. Pictures taken under 10X magnification were blindly quantified using ImageJ software.

Electrophoresis:

Article Title: Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription
Article Snippet: For the super shift reaction 1 µg of anti-RARα (C-20, Santa Cruz) or anti-RXR (F-1, Santa Cruz) antibodies were included to the reaction just before adding the Biotin labeled oligos ( Supplementary Table S2 ). .. Binding reactions were incubated at room temperature for 40 min and then resolved on a 6% native polyacrylamide gel in 0.5× TBE in a minigel electrophoresis apparatus (dimensions 8 × 8 × 0.1 cm).

Negative Control:

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz). .. IgG was used as a negative control for the qChIP assays.

Article Title: Regulation of cyp26a1 on Th17 cells in mouse peri-implantation
Article Snippet: Normal rat IgG was used as a negative control. .. Here is the primary and secondary antibodies list used in immunohistochemistry: anti-RORγt (17-6988, eBioscience, USA), anti-IL-17 (sc-7929, Santa Cruz,USA), anti-cyp26a1 (CYP26A12-A, Acris, Hiddenhausen, Germany), anti-RARα (sc-15040, Santa Cruz, USA); goat anti-rat (112-035-003, Jackson ImmunoResaerch, USA), goat anti-rabbit (074-1506, KPL, USA), rabbit anti-goat (14-13-06, KPL, USA).

Recombinant:

Article Title: Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling
Article Snippet: Recombinant human tumor necrosis factor (TNFα) was obtained from Peprotech (Rocky Hill, NJ, USA). .. Antibodies recognizing p65, IκBα, and RARα were purchased from Santa Cruz Biotechnology.

Article Title: Retinoic Acid Activates Monoamine Oxidase B Promoter in Human Neuronal Cells *
Article Snippet: Monoclonal anti-RXRα (sc-46659), anti-Sp1 (sc-17824), anti-β-actin (sc-47778), anti-GST (sc-138), polyclonal anti-RARα (sc-773), anti-RARα (sc-551), anti-RXRα (sc-774), and anti-GFP (sc-8334) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). .. Mouse TrueBlotTM horseradish peroxidase (HRP) anti-mouse IgG (13-8817) was purchased from eBioscience, Inc. Human Sp1 recombinant protein was purchased from Promega (Madison, WI).

Protein Binding:

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *
Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz). .. The level of endogenous Sp1 and p53 protein binding was analyzed using polyclonal antibodies against anti-p53 (Santa Cruz), anti-Sp1 (Santa Cruz), anti-Ac-Histone 3, anti-Ac-Histone 4, anti-Histone H3K4-Me3, and anti-Histone H3K9-Me3 (Upstate).

Concentration Assay:

Article Title: Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling
Article Snippet: When cells were treated with these reagents, matching concentrations of vehicle were used as the control and the final concentration of DMSO was kept at or below 0.1% in all experiments. .. Antibodies recognizing p65, IκBα, and RARα were purchased from Santa Cruz Biotechnology.

End Labeling:

Article Title: Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription
Article Snippet: Oligonucleotide labeling was performed using the Biotin 3′ End Labeling Kit (Pierce Biotechnology). .. For the super shift reaction 1 µg of anti-RARα (C-20, Santa Cruz) or anti-RXR (F-1, Santa Cruz) antibodies were included to the reaction just before adding the Biotin labeled oligos ( Supplementary Table S2 ).

Lysis:

Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression
Article Snippet: For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA). .. Briefly, 400 μg of protein in 100 μl of lysis buffer was incubated with the selected antibodies overnight at 4 °C with rotation.

Article Title: Targeting the Acute Promyelocytic Leukemia-Associated Fusion Proteins PML/RAR? and PLZF/RAR? with Interfering Peptides
Article Snippet: Cell lysates were prepared in an SDS lysis buffer (1.5 M Tris-HCl, pH 6.8, 20% SDS, 10% Glycerol). .. Western blot analysis was performed using the anti-RARα (C-20 - Santa Cruz Biotechnology, Santa Cruz, USA), anti-GFP (FL - St. Cruz), anti-c-ABL (24-11, St. Cruz Biotechnology, Santa Cruz, USA) and anti-phospho-ABL (Tyr245, Millipore, Billerica, USA), anti-GAPDH (FL-335 - St. Cruz), and anti-tubulin (Ab-4, NeoMarkers, Thermo Scientific Inc., Kalamazoo, USA) antibodies.

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  • 79
    Santa Cruz Biotechnology antibodies against rarα
    IRAK-1 and Tollip are involved in the low dose LPS mediated suppression of <t>RARα</t>
    Antibodies Against Rarα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against rarα/product/Santa Cruz Biotechnology
    Average 79 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    antibodies against rarα - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    76
    Santa Cruz Biotechnology antibodies primary antibodies rarα
    Effects of arsenicals on differentiation of NB4 cells and <t>PML-RARα</t> fusion protein degradation NB4 cells were exposed to 1 μM of arsenicals for 24 and 72 h to determine the changes in PRAM-1 protein A. and quantified by Image J public domain software B. Additionally, NB4 cells were stained with Wright-Giemsa C. and the expression of CD11b in NB4 cells were determined by flow cytometry D. Degradation of PML-RARα fusion protein in NB4 cells were determined by western blot with RARα antibody E. For immunofluorescence, NB4 cells were double-labeled with PML (red) and SUMO-1 (green) after exposure to 1 μM arsenicals for 12 h. Blue fluorescence indicates cell nucleus. Cells were imaged with a laser scanning confocal microscope F. ATRA was used as positive control in the present study. Asterisks indicate a significant difference from the untreated control group at * p
    Antibodies Primary Antibodies Rarα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies primary antibodies rarα/product/Santa Cruz Biotechnology
    Average 76 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies primary antibodies rarα - by Bioz Stars, 2020-02
    76/100 stars
      Buy from Supplier

    Image Search Results


    IRAK-1 and Tollip are involved in the low dose LPS mediated suppression of RARα

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    Article Title: Molecular Mechanisms responsible for the reduced expression of cholesterol transporters from macrophages by low dose endotoxin

    doi: 10.1161/ATVBAHA.112.300049

    Figure Lengend Snippet: IRAK-1 and Tollip are involved in the low dose LPS mediated suppression of RARα

    Article Snippet: The antibodies against RARα, GAPDH, SRC3, GSK3β, pGSK3β-Tyr216 and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques:

    Increase in RARα at uterine implantation sites following pCR3.1-cyp26a1 immunization during mice peri-implantation and further certainty, the influence of RARα on Th17 cells at uterine implantation sites by tail vein injection of RARα specific agonist and antagonist during mice peri-implantation. (A) RARα levels were significantly increased at uterine implantation sites based on Western blotting. β-actin was used as a loading control. (B) RARα levels were obviously increased at implantation sites of D5 pregnancy based on immunohistochemistry following pCR3.1-cyp26a1 immunization; bars = 200 and 25 μm. The data were derived from three separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. E, embryo; S, uterine stroma; LE, Luminal epithelium; GE, Glandular epithelium. (C) RARα and RORγt protein expression changes in uterus on D5 of pregnancy in response to administration of agonist (Am-580) and antagonist (Ro-41-5253) of RARα. RARα levels were significantly increased by the activation of Am-580 and significantly reduced by the inhibition of Ro-41-5253 at uterine implantation sites based on western blotting of pregnancy D5. RORγt levels just took the opposite changes on RARα. β-actin was used as a loading control. The administration (125 μg/mouse) of RARα agonist (Am-580) or antagonist (Ro-41-5253) was performed on D3 of pregnancy through tail vein injection; ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Regulation of cyp26a1 on Th17 cells in mouse peri-implantation

    doi: 10.1111/jcmm.12196

    Figure Lengend Snippet: Increase in RARα at uterine implantation sites following pCR3.1-cyp26a1 immunization during mice peri-implantation and further certainty, the influence of RARα on Th17 cells at uterine implantation sites by tail vein injection of RARα specific agonist and antagonist during mice peri-implantation. (A) RARα levels were significantly increased at uterine implantation sites based on Western blotting. β-actin was used as a loading control. (B) RARα levels were obviously increased at implantation sites of D5 pregnancy based on immunohistochemistry following pCR3.1-cyp26a1 immunization; bars = 200 and 25 μm. The data were derived from three separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. E, embryo; S, uterine stroma; LE, Luminal epithelium; GE, Glandular epithelium. (C) RARα and RORγt protein expression changes in uterus on D5 of pregnancy in response to administration of agonist (Am-580) and antagonist (Ro-41-5253) of RARα. RARα levels were significantly increased by the activation of Am-580 and significantly reduced by the inhibition of Ro-41-5253 at uterine implantation sites based on western blotting of pregnancy D5. RORγt levels just took the opposite changes on RARα. β-actin was used as a loading control. The administration (125 μg/mouse) of RARα agonist (Am-580) or antagonist (Ro-41-5253) was performed on D3 of pregnancy through tail vein injection; ** P

    Article Snippet: Here is the primary and secondary antibodies list used in immunohistochemistry: anti-RORγt (17-6988, eBioscience, USA), anti-IL-17 (sc-7929, Santa Cruz,USA), anti-cyp26a1 (CYP26A12-A, Acris, Hiddenhausen, Germany), anti-RARα (sc-15040, Santa Cruz, USA); goat anti-rat (112-035-003, Jackson ImmunoResaerch, USA), goat anti-rabbit (074-1506, KPL, USA), rabbit anti-goat (14-13-06, KPL, USA).

    Techniques: Mouse Assay, Injection, Western Blot, Immunohistochemistry, Derivative Assay, Expressing, Activation Assay, Inhibition

    PPARβ/δ suppresses MMP2 activity by interfering with RAR signaling A. A putative RAR RE in the human MMP2 promoter. +1 represented the transcriptional start site. B. Representative EMSA of nuclear extracts from NT2/D1-MigR1 (MigR1) and NT2/D1-hPPARβ/δ (hPPARβ/δ) cells were incubated with either double-stranded oligonucleotides (oligo) encoding the RAR RE MMP2 promoter or mutated oligonucleotides (mutant oligo). Black arrowheads indicated the presence of oligonucleotide-protein complexes. White arrowheads indicated the super shift of oligonucleotide-protein-anti-RAR-antibody complexes. C. Representative photomicrograph of the ChIP assay for RAR occupancy on the MMP2 promoter. D. RARα protein expression in NT2/D1, MigR1 and hPPARβ/δ cells transiently transfected with pSG5-RARα plasmid. E, F. CYP26A1 and MMP2 mRNA expression in MigR1 and hPPARβ/δ cells or cells transiently over-expressing RARα after atRA treatment, respectively. G. Left panel, activities of MMP2 and MMP9 in MigR1 and hPPARβ/δ cells transiently over-expressing RAR after atRA treatment, right panel, relative activity of pro-MMP2 in MigR1 and hPPARβ/δ cells. H. ChIP-qPCR showing effect of PPARβ/δ on RARα occupancy on MMP2 promoter in cells over-expressing PPARβ/δ or RARα following atRA and/or GW0742 treatment. Values represent mean ± S.E.M. Values with different superscript letters are significantly different at p ≤ 0.05.

    Journal: Oncotarget

    Article Title: Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms

    doi:

    Figure Lengend Snippet: PPARβ/δ suppresses MMP2 activity by interfering with RAR signaling A. A putative RAR RE in the human MMP2 promoter. +1 represented the transcriptional start site. B. Representative EMSA of nuclear extracts from NT2/D1-MigR1 (MigR1) and NT2/D1-hPPARβ/δ (hPPARβ/δ) cells were incubated with either double-stranded oligonucleotides (oligo) encoding the RAR RE MMP2 promoter or mutated oligonucleotides (mutant oligo). Black arrowheads indicated the presence of oligonucleotide-protein complexes. White arrowheads indicated the super shift of oligonucleotide-protein-anti-RAR-antibody complexes. C. Representative photomicrograph of the ChIP assay for RAR occupancy on the MMP2 promoter. D. RARα protein expression in NT2/D1, MigR1 and hPPARβ/δ cells transiently transfected with pSG5-RARα plasmid. E, F. CYP26A1 and MMP2 mRNA expression in MigR1 and hPPARβ/δ cells or cells transiently over-expressing RARα after atRA treatment, respectively. G. Left panel, activities of MMP2 and MMP9 in MigR1 and hPPARβ/δ cells transiently over-expressing RAR after atRA treatment, right panel, relative activity of pro-MMP2 in MigR1 and hPPARβ/δ cells. H. ChIP-qPCR showing effect of PPARβ/δ on RARα occupancy on MMP2 promoter in cells over-expressing PPARβ/δ or RARα following atRA and/or GW0742 treatment. Values represent mean ± S.E.M. Values with different superscript letters are significantly different at p ≤ 0.05.

    Article Snippet: Antibodies against RARα, progesterone receptor (PR), and normal rabbit-IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were individually added to histone-DNA complexes for immunoprecipitation of protein bound chromatin.

    Techniques: Activity Assay, Incubation, Mutagenesis, Chromatin Immunoprecipitation, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    PPARβ/δ interferes with atRA-stimulated signaling in NT2/D1 cells A. Quantitative western blot analysis of RXRα, RARα, OCT3/4, FABP5 and CRABPII expression in NT2/D1, NT2/D1-MigR1 (MigR1) and NT2/D1-hPPARβ/δ (hPPARβ/δ) cells. B, C. Quantitative western blot analysis of RXRα, RARα, CYP26A1, FABP5 and CRABPII expression in NT2/D1, MigR1 and hPPARβ/δ cells in response to atRA treatment. Values represent mean ± S.E.M. Values with different superscript letters are significantly different at p ≤ 0.05.

    Journal: Oncotarget

    Article Title: Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms

    doi:

    Figure Lengend Snippet: PPARβ/δ interferes with atRA-stimulated signaling in NT2/D1 cells A. Quantitative western blot analysis of RXRα, RARα, OCT3/4, FABP5 and CRABPII expression in NT2/D1, NT2/D1-MigR1 (MigR1) and NT2/D1-hPPARβ/δ (hPPARβ/δ) cells. B, C. Quantitative western blot analysis of RXRα, RARα, CYP26A1, FABP5 and CRABPII expression in NT2/D1, MigR1 and hPPARβ/δ cells in response to atRA treatment. Values represent mean ± S.E.M. Values with different superscript letters are significantly different at p ≤ 0.05.

    Article Snippet: Antibodies against RARα, progesterone receptor (PR), and normal rabbit-IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were individually added to histone-DNA complexes for immunoprecipitation of protein bound chromatin.

    Techniques: Western Blot, Expressing

    Effects of arsenicals on differentiation of NB4 cells and PML-RARα fusion protein degradation NB4 cells were exposed to 1 μM of arsenicals for 24 and 72 h to determine the changes in PRAM-1 protein A. and quantified by Image J public domain software B. Additionally, NB4 cells were stained with Wright-Giemsa C. and the expression of CD11b in NB4 cells were determined by flow cytometry D. Degradation of PML-RARα fusion protein in NB4 cells were determined by western blot with RARα antibody E. For immunofluorescence, NB4 cells were double-labeled with PML (red) and SUMO-1 (green) after exposure to 1 μM arsenicals for 12 h. Blue fluorescence indicates cell nucleus. Cells were imaged with a laser scanning confocal microscope F. ATRA was used as positive control in the present study. Asterisks indicate a significant difference from the untreated control group at * p

    Journal: Oncotarget

    Article Title: Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation

    doi:

    Figure Lengend Snippet: Effects of arsenicals on differentiation of NB4 cells and PML-RARα fusion protein degradation NB4 cells were exposed to 1 μM of arsenicals for 24 and 72 h to determine the changes in PRAM-1 protein A. and quantified by Image J public domain software B. Additionally, NB4 cells were stained with Wright-Giemsa C. and the expression of CD11b in NB4 cells were determined by flow cytometry D. Degradation of PML-RARα fusion protein in NB4 cells were determined by western blot with RARα antibody E. For immunofluorescence, NB4 cells were double-labeled with PML (red) and SUMO-1 (green) after exposure to 1 μM arsenicals for 12 h. Blue fluorescence indicates cell nucleus. Cells were imaged with a laser scanning confocal microscope F. ATRA was used as positive control in the present study. Asterisks indicate a significant difference from the untreated control group at * p

    Article Snippet: Antibodies Primary antibodies RARα, PRAM-1, rabbit anti-Poly (ADP-ribose) polymerase (PARP) polyclonal antibody, Cyt c, JNK, P-JNK, P-PERK, eif2α, rabbit anti-human PML, anti-human SUMO-1, Ub Antibody were purchased from Santa Cruz Biotechnology (CA, USA).

    Techniques: Software, Staining, Expressing, Flow Cytometry, Cytometry, Western Blot, Immunofluorescence, Labeling, Fluorescence, Microscopy, Positive Control

    Examination of PML-NBs formation or PML and PML-RARα proteins degradation in HEK293T and HeLa cells after exposure to arsenicals Degradation of PML-RARα fusion protein was examined in PML-RARα -transfected HeLa cells following exposure to 4 μM of arsenicals at indicated time points A. Likewise, degradation of PML protein was determined in PML -HEK293T cells following exposure to 4 μM of arsenicals for 6 h B. Formation of PML-NBs in PML or PML-RARα overexpressed HEK293T C. or HeLa cells D. were determined after exposure to arsenicals for 6 h. Cells were double-labeled with PML (green) and SUMO-1 (red). Blue fluorescence indicates cell nucleus. Arrow indicates the PML-NBs.

    Journal: Oncotarget

    Article Title: Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation

    doi:

    Figure Lengend Snippet: Examination of PML-NBs formation or PML and PML-RARα proteins degradation in HEK293T and HeLa cells after exposure to arsenicals Degradation of PML-RARα fusion protein was examined in PML-RARα -transfected HeLa cells following exposure to 4 μM of arsenicals at indicated time points A. Likewise, degradation of PML protein was determined in PML -HEK293T cells following exposure to 4 μM of arsenicals for 6 h B. Formation of PML-NBs in PML or PML-RARα overexpressed HEK293T C. or HeLa cells D. were determined after exposure to arsenicals for 6 h. Cells were double-labeled with PML (green) and SUMO-1 (red). Blue fluorescence indicates cell nucleus. Arrow indicates the PML-NBs.

    Article Snippet: Antibodies Primary antibodies RARα, PRAM-1, rabbit anti-Poly (ADP-ribose) polymerase (PARP) polyclonal antibody, Cyt c, JNK, P-JNK, P-PERK, eif2α, rabbit anti-human PML, anti-human SUMO-1, Ub Antibody were purchased from Santa Cruz Biotechnology (CA, USA).

    Techniques: Transfection, Labeling, Fluorescence