transam transcription factor assay kits for sp1/sp3  (Active Motif)

Journal: Free radical biology & medicine

Article Title: The HIV-Tat protein interacts with Sp3 transcription factor and inhibits its binding to a distal site of the sod2 promoter in human pulmonary artery endothelial cells

doi: 10.1016/j.freeradbiomed.2019.12.015

Figure Lengend Snippet: Using an Sp-specific oligonucleotide-binding ELISA (TransAm, Active Motif), we measured the levels of active Sp1 (Panel A) and Sp3 (Panel B) in the nucleus of mock transfected (Tat(−)) or pCP2-Tat101 transfected (Tat(+)) HPAEC. The signal was normalized to μg of nuclear protein. Mean ± SEM of biological replicates, n = 3, (***p ≤ 0.001 by unpaired student’s T-test).

Article Snippet: DNA-binding ELISAs for active Sp1 and Sp3 were carried out on nuclear extracts prepared as previously described [ 21 ] using Sp1 and Sp3-specific TransAM kits (Active Motif) according to manufacturer’s protocol.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Conserved Sp1 response elements in TATA-less Prdx6 promoter of mouse, rat and human.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Binding Assay

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Aging human LECs/lenses showing elevated levels of ROS and progressive decline in Sp1 and Prdx6 expression connected to reduction in Sp1 activity. ( A ) ROS levels increased progressively in aging hLECs. Cells were cultured in 96 well plate (5000/well), and ROS were quantified using H2-DCF-DA dye assay as shown. Data represent the mean ± S.D. of two independent experiments. Younger (18y) vs aging samples; * p < 0.001. ( B ) Aging hLECs displayed progressive decline in levels of Sp1 and its target gene, Prdx6 mRNA. Total RNA was isolated from human LECs/ lenses of different ages as indicated and was processed for real-time PCR analysis with specific primers. The data represent the mean ± S.D. from three independent experiments. p values were determined for younger vs aging samples. * p < 0.001. ( C ) Aging/aged human lenses/LECs displayed significant loss of Sp1 activity. Nuclear extracts prepared from aging/aged hLECs/lenses were used for assay. LECs/lenses were divided into five age groups: 16-18y (n=6); 21-26y (n=6); 52-58y (n=8); 62-66y (n=8); 74-76y (n=8). Nuclear extracts containing equal amounts of protein were processed and assayed for Sp1 activity using a commercially available kit (Active motif) as described in Materials and Methods. The data represent the mean ± S.D. from three independent experiments. p values were determined for younger vs aging samples. * p < 0.001. ( D ) Nuclear extracts containing equal amounts of proteins were processed for Sandwich ELISA to measure the total Sp1 protein. Total Sp1 proteins were equalized with the O.D. of Sandwich ELISA and processed for Sp1 transactivation assay using a commercially available kit (Active motif) as ascribed in Materials and Methods. P value were determined for younger vs aging samples. * p < 0.001. ( E ) Aging hLECs showing significant loss of Sp1 protein. Cellular proteins were isolated from hLECs and human lenses of different ages as described in Materials and Methods section, and as indicated. An equal amount of protein was loaded onto SDS-PAGE, and immunoblotted using Sp1 antibody. Upper panel; expression levels of Sp1, Lower panel; membrane probed with β-actin antibody as loading/internal control. Each band of blot was quantified using densitometer shown below. Images are representatives from three independent experiments. P value were determined for younger vs aging samples. * p < 0.001.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Sandwich ELISA, Transactivation Assay, SDS Page, Membrane, Control

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: ChIP analysis of genomic DNA from LECs facing oxidative stress disclosed a significant loss in Sp1-DNA binding to Prdx6 gene promoter. ( A ) Schematic illustration of 5’-proximal promoter region of Prdx6 containing Sp1 binding sites showing primer location and sequences used in ChIP assay. ( B and C ) ChIP assay showing Sp1 binding to Prdx6 promoter in vivo . Chromatin samples were prepared from Prdx6 +/+ LECs (mLECs) exposed to different doses of H 2 O 2 (0, 25, 50 and 75µM) and/or UVB (0, 30, 60 and 90J/m 2 ) as indicated. 72h later samples were subjected to ChIP assay with ChIP grade antibodies, anti-Sp1 or IgG control. The DNA fragments were amplified by using primers designed to amplify −208 to +27 region of the Prdx6 promoter bearing Sp1 sites (**) and contiguous sequence (−2229 to −2356) to which Sp1 does not bind (*) as indicated. PCR products were resolved onto agarose gel and visualized with ethidium bromide staining. Photographs are representative of three experiments. ( D and E ) Expression assays showing H 2 O 2 - and UVB- induced declined expression of Sp1 in mLECs. mLECs cells were treated with different concentrations of H 2 O 2 ( D ) and/or UVB ( E ) multiple time for 3 days as indicated. Total RNA and protein were isolated and subjected to real-time PCR and Western analysis with Sp1 specific probes, respectively. Data revealed a concentration –dependent reduced expression of Sp1 mRNA ( Da and Ea ; Gray vs black bars; * p <0.001) and protein ( Db and Eb ).

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Binding Assay, In Vivo, Control, Amplification, Sequencing, Agarose Gel Electrophoresis, Staining, Expressing, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Oxidative stress attenuated Sp1 binding to its GC-Box elements present in hPrdx6 gene promoter. . Evolutionary conserved Sp1 binding sequences in TATA-less Prdx6 promoters of mouse, rat and human cells. ( A ) Schematic illustration of 5’-proximal promoter region of Prdx6 containing Sp1 (GC-Box) binding sites showing primer location and sequences used in ChIP assay. ( B and C ) Oxidative stress (H 2 O 2 or UVB)-induced reduction in DNA binding activity of Sp1 to hPrdx6 gene promoter containing GC-Box (Sp1 sites) in SRA-hLECs. ChIP assay was carried out by using ChIP-IT® Express and ChIP-IT® qPCR analysis kits (Active motif). Chromatin samples prepared from SRA-hLECs were exposed to varying concentrations of H 2 O 2 (0, 50, 75 and 100µM) or UVB (0, 40, 80 and 120J/m 2 ), and were subjected to ChIP assay with ChIP grade antibodies, anti-Sp1 (black bars) and control IgG (gray bars). The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 gene promoter bearing GC-box (Sp1 sites). Histogram showed the amplified DNA by qPCR analysis: ( B ) Control (0) vs 50µM vs 75µM vs 100µM H 2 O 2 treatment. ( C ) Control (0) vs 40J/m 2 vs 80J/m 2 vs 120J/m 2 UVB exposure. The data represent mean ± SD from three independent experiments (** p <0.05; * p <0.001). ( D ) Human Prdx6 promoter activity inhibited by mithramycin A (Mithra A), an inhibitor of Sp1, validated Sp1 regulation of hPrdx6 gene. Cells were transfected with CAT-hPrdx6 (-918/+30) or empty CAT vector construct and treated with Mithra-A at different concentrations for 24h. Data represent mean ± SD from three independent experiments (** p <0.05; * p <0.001).

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Binding Assay, Activity Assay, Control, Amplification, Transfection, Plasmid Preparation, Construct

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Prdx6 -/- LECs, a model for aging, bore an enhanced Sumoylated form of Sp1, and levels were further increased with exposure to oxidative stress. ( A ) Nuclear extracts were prepared from Prdx6 +/+ and Prdx6 −/− mLECs and submitted to Sp1 Sandwich/Sumo1-ELISA assays to examine the total and Sumoylated forms of Sp1 protein. Sumoylated Sp1 protein was subtracted from total Sp1 protein, and results are presented as deSumoylated (gray bars) and Sumoylated (black bars) forms of Sp1. The data represent mean ± SD from three independent experiments. Prdx6 +/+ vs Prdx6 −/− ; * p <0.001. ( B - E ) Prdx6 -/- LECs displayed increased levels of Sumoylated Sp1 in response to increased oxidative loads. Prdx6 -/- LECs were exposed to H 2 O 2 ( B and C ) or UVB ( D and E ) in different concentrations and for different time intervals as indicated. Nuclear extracts were prepared and used to perform Sp1 sandwich/Sumo1-ELISA specific to Sp1. Sumoylated Sp1 protein was subtracted from total Sp1 protein, presented as deSumoylated Sp1 (gray bars) and Sumoylated Sp1 (black bars). B-E: Data represent mean ± SD from three independent experiments. 0µM vs 25, 50 and 75µM (H 2 O 2 ), 0 min vs 15 , 30 and 90 min of H 2 O 2 exposure and 0 J/m 2 vs 30, 60 and 90 J/m 2 (UVB) and 0h vs 3, 6 and 12h UVB exposure; ** p <0.05; * p <0.001.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Aging human LECs/lenses showed age-dependent decline in Senp1 expression and increased Sumo1 expression, which were directly related to increase Sp1 Sumoylation. ( A ) Aging hLECs/lenses displayed a progressive decline in the deSumoylating agent Senp1 and an increase in Sumo1 levels. Total RNA was isolated from human lenses/LECs of different ages as indicated, and was processed for real-time qPCR analysis as stated in Materials and Methods. The data represent the mean ± S.D. values from three independent experiments. P values were determined for younger (18y) vs aging samples. ** p <0.05, * p < 0.001. ( B ) Nuclear lysates were prepared from hLECs/lenses of various ages and were submitted to Sp1 sandwich/Sumo1-ELISA to examine the total and Sumoylated Sp1 protein. Primary hLECs isolated from lenses of different ages were divided into four groups: 16-18y (n=6); 52-58y (n=8); 62-66 (n=8); and 74-76y (n=8). Sumoylated Sp1 protein was subtracted from total Sp1 protein, and presented as deSumoylated (B, gray bars) and Sumoylated (B, black bars) forms of Sp1. Data represent mean ± SD from two independent experiments. p values were determined for younger (16-18y) vs aging samples. * p < 0.001.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Cells overexpressing Sumo1 showed reduced Sp1 binding to its responsive elements in Prdx6 promoter. ( A ) Schematic illustration of Prdx6 gene promoter. ( B ) Gel-shift mobility assay showing that Sumo1 reduced the Sp1 DNA–binding activity of Prdx6 gene promoter. Gel-shift mobility assay was carried out using nuclear extracts isolated from mLECs transfected with pEGFP-Vector (Lanes 1 and 2) or pEGFP-Sumo1 (Lanes 3 and 4) incubated with 32p-labeled wild type probe (Lanes 1 and 3) or its mutant (Lanes 2 and 4). A diminished Cm1 band was observed in cells overexpressing Sumo1 (Lane 3) in comparison to vector control (Lane 1). No binding occurred in mutant probes (Lanes 2 and 4). ( C ) Histogram represents densitometry analysis of DNA-protein complex formed in gel-shift assay. Lane 1 vs lane 3, * p < 0.001. ( D ) ChIP assay showing Sumo1 overexpression significantly suppressed Sp1-DNA binding in Prdx6 gene promoter in dose-dependent manner. mLECs were transiently transfected with different concentrations of pEGFP-Sumo1 (0, 1, 2 and 4 μg). 72h later ChIP assay was carried out with anti-Sp1 and control IgG antibodies. Pulled DNA fragments were subjected to PCR analysis for Sp1 binding cis -elements of Prdx6 promoter. The product was analyzed through agarose-gel. Data represent three experiments. ( E ) Senp1 overexpression dramatically enhanced Sp1-DNA binding in concentration-dependent -manner. mLECs were transfected with increasing concentrations of pFlag-Senp1 (0, 0.5, 1 and 2μg) for 72h. ChIP analysis was carried out using chromatin samples prepared from transfected LECs with a ChIP grade antibody, anti-Sp1 and control IgG. The DNA fragments were used as templates for PCR by using primers designed to amplify −208 to +27 region of the Prdx6 promoter bearing Sp1 binding sites as shown. PCR product was analyzed through agarose gel as shown.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Binding Assay, Gel Shift, Activity Assay, Isolation, Transfection, Plasmid Preparation, Incubation, Labeling, Mutagenesis, Comparison, Control, Over Expression, Agarose Gel Electrophoresis, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Sumo1 modified Sp1 at K16 residue in vivo . ( A ) SRA-hLECs (1.2X10 6 ) were transfected with pEGFP-Sumo1 (3µg) along with Sp1 WT (3µg) or its mutant Sp1K16R (mutated at Sumoylation sites) using (3µg) plasmid as indicated. Exogenous Sp1 was immunoprecipitated (IP) from cell lysates containing equal amounts of proteins, and its Sumoylated form was detected with anti-HA ( Ba ) and anti-Sumo1 ( Bb ) rabbit polyclonal antibodies as indicated. Cell lysates were prepared and subjected to IP using anti-HA monoclonal antibody. IP with anti-HA monoclonal antibody shows a single-exogenous Sumoylated band at ~145 kDa (lane 2, pEGFP-Sumo1+pCl-neo-HA-Sp1WT). No Sumoylation band could be detected in cell extracts of pEGFP-Vector+pCl-neo-HA-Sp1WT or pEGFP-Sumo1+pCl-neo-HA-Sp1-K16R transfected cells ( B , a and b ; lanes 1 and 3). ( B ) SRA-hLECs were transfected with pCl-neo-HA-Sp1WT plus pEGFP-Vector, or pCl-neo-HA-Sp1WT plus pEGFP-Sumo1, or pCl-neo-HA-Sp1-K16R plus pEGFP-Sumo1. 48h later, total cell lysates were prepared and processed for Sumo1-ELISA assay according to the manufacturer’s protocol (EpiQuik TM ) to measure the Sumoylated form of Sp1. Data represent mean ± SD from three independent experiments: pCl-neo-HA-Sp1WT plus pEGFP-Vector, vs pCl-neo-HA-Sp1WT plus pEGFP-Sumo1, vs pCl-neo-HA-Sp1 K16R plus pEGFP-Sumo1 (* p <0.001).

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Modification, Residue, In Vivo, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Mutagenesis and in vivo DNA binding assay revealed increased binding of Sumoylation-deficient Sp1K16R to Sp1 site in Prdx6 promoter by skipping aberrant Sumoylation effect. ( A ) Schematic representation of the regulatory region of proximal promoter of Prdx6 gene containing GC-box (Sp1 binding sites) showing primer location used in ChIP assay. ( B ) Sumo1 failed to affect Sp1-DNA binding activity in Sumoylation-deficient Sp1K16R transfected LECs. SRA-hLECs were transfected with either pCl-neo-HA-Sp1 or its mutant pCl-neo-HA-Sp1K16R alone or cotransfected with different concentrations of pEGFP-Sumo1. ChIP experiment was carried out as described in Materials and Methods. Chromatin samples prepared from LECs cotransfected with pEGFP-Sumo1 with either pCl-neo-HA-Sp1 or its mutant pCl-neo-HA-Sp1K16R were subjected to ChIP assay with a ChIP grade antibody, anti-HA (gray and black bars) and control IgG (open bars). The DNA fragments were used as templates for RT-qPCR by using primers designed to amplify −342 to +30 region of the Prdx6 gene promoter bearing Sp1 binding sites as shown. Histogram shows the amplified DNA through real-time qPCR analysis. 0 µg vs 2 µg and 4µg pEGFP-Sumo1, pCl-neo-HA-Sp1 WT vs pCl-neo-HA-Sp1K16R (* p < 0.001). ( C ) Senp1 overexpression showed increased Sp1 DNA binding of Sp1WT and comparable to Sumoylation deficient Sp1K16R. SRA-hLECs were cotransfected with pFlag-Senp1 with either pCl-neo-HA-Sp1 or mutant pCl-neo-HA-Sp1K16R as indicated. ChIP assay was conducted as described above using anti-HA antibody. Histograms represent the concentration dependence of Senp1-induced enrichment of Sp1 at its binding sites in Prdx6 gene promoter. 0 µg vs 0.5 µg and 1µg pFlag-Senp1, pCl-neo-HA-Sp1 WT vs pCl-neo-HA-Sp1K16R (** p <0.05; * p < 0.001).

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Mutagenesis, In Vivo, DNA Binding Assay, Binding Assay, Activity Assay, Transfection, Control, Quantitative RT-PCR, Amplification, Over Expression, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Sp1 (K16R) mutated at Sumoylation site enhanced its transcription potential by increasing steady state of Sp1 in cells. ( A ) SRA-hLECs were cotransfected with wild type Prdx6 promoter linked to CAT along with either pCl-neo-HA-Sp1 or pCl-neo-HA-Sp1K16R as shown. After 72h cell lysates were analyzed for CAT activity. Histograms represent values derived from three independent experiments. * p < 0.001. ( B ) Relative protein stability of Sp1WT vs Sumoylation-deficient mutant Sp1K16R. SRA-hLECs were transiently transfected with pCl-neo-HA-Sp1WT or its mutant, pCl-neo-HA-Sp1K16R. After 36h, the transfectants were treated with different concentrations of CHX (10 and 20µg/ml) for 24h ( Ba ) or CHX (10 and 20µg/ml) for 36h ( Bb ) or CHX (10 and 20µg/ml) for 48h ( Bc ) or CHX (40µg/ml) for 8h ( Bd ) or as indicated. Total lysates with equal amounts of proteins were western blotted (WB) with anti-HA antibody. Anti-β-actin or anti-Tubulin antibodies were used as loading control. The percentage of remaining Sp1 (Sp1WT and its mutant Sp1 K16R) protein after the CHX translational inhibitor treatment is presented as histogram in right side of Western blot based upon densitometry quantitation. Control vehicle (DMSO) vs CHX treated, ** p <0.05; * p <0.001.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Activity Assay, Derivative Assay, Mutagenesis, Transfection, Western Blot, Control, Quantitation Assay

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Sp1K16R mutated at Sumo1 binding site provided enhanced cytoprotection against oxidative stress. ( A and B ) SRA-hLECs were transfected with either pEGFP-Vector, pCl-neo-HA-Sp1, or pCl-neo-HA-Sp1K16R and then exposed to different concentrations of H 2 O 2 as indicated. After 8h of H 2 O 2 exposure, ROS intensity was quantified with CellROX deep red reagent ( A ). 24h later viability of cells was analyzed by MTS assay ( B ) as shown. Histogram values represent mean ± SD of three independent experiments. 0 vs 100 vs 150µM H 2 O 2 and pEGFP-Vector vs pCl-neo-HA-Sp1 WT vs pCl-neo-HA-Sp1K16R (** p <0.05; * p <0.001). ( C and D ) SRA-hLECs were transfected with pEGFP-Sumo1 along with either pCMV-Vector (open bars), pCl-neo-HA-Sp1 (gray bars), or pCl-neo-HA-Sp1K16R (black bars), and then exposed to oxidative stress. ROS intensity ( C ) and cell viability ( D ) are presented as histograms. Values represent mean ± SD of three independent experiments. 0 vs 100µM H 2 O 2 and pEGFP-Sumo1 vs pEGFP-Sumo1 plus pCl-neo-HA-Sp1 WT vs pEGFP-Sumo1 plus pCl-neo-HA-Sp1K16R (** p <0.05; * p <0.001). Sumoylation-deficient Sp1K16R (black bars) showed significantly higher protection and reduced ROS production, indicating that mutant Sp1K16R was more effective in protecting cells from oxidative stress Sumoylation-mediated insults.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Binding Assay, Transfection, Plasmid Preparation, MTS Assay, Mutagenesis

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Sumoylation-deficient Prdx6K122/142R fused to transduction protein domain (TAT) internalized in cells and blunted oxidative stress-induced aberrant Sumoylation. ( A and B ) Prdx6 -/- mLECs were transduced with Sumoylation-deficient protein, TAT-HA-Prdx6K122/142R conferred higher resistance to oxidative stress-induced Sumoylation than did Prdx6WT. Prdx6 -/- LECs were pretreated with TAT-HA-Prdx6 WT or TAT-HA-Prdx6K122/142R and then exposed to different concentrations of H 2 O 2 (0, 25, 50 and 75µM) and/or UVB (0, 30, 60 and 90J/m 2 ). 48h later, nuclear extracts containing equal amounts of proteins were processed for Sumo1-ELISA assay to assess the relative levels of Sp1 Sumoylation in Prdx6 WT (gray bars) and its mutant Prdx6K122/142R (black bars) transduced in cells as shown. Data represent the mean ± SD from three independent experiments (** p <0.05, * p <0.001). ( C ) Transduction of TAT-HA-Prdx6 and TAT-HA-Prdx6K122/142R into cells. An aliquot of 10 μg/ml recombinant protein was added to culture media and transduction of TAT-HA-Prdx6 (Lane 3) and TAT-HA-Prdx6K122/142 R (Lane 4) was assessed using WB by anti-Prdx6 antibody. ( D ) Represents the TAT-HA-Prdx6 and TAT-HA-Prdx6K122/142R following H 2 O 2 and/or UVB oxidative exposure treatment schedule.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Transduction, Enzyme-linked Immunosorbent Assay, Mutagenesis, Recombinant

Journal: Aging (Albany NY)

Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

doi: 10.18632/aging.101547

Figure Lengend Snippet: Enhanced Sp1 binding to Prdx6 promoter in cells transduced with Sumoylation–deficient Prdx6 against oxidative stress. ( A and B ) SRA-hLECs were transduced with TAT-HA-Prdx6WT and its mutant TAT-HA-Prdx6K122/142R mutated at Sumoylation sites recombinant protein followed by different concentrations of H 2 O 2 ( A ) or UVB ( B ) exposure as indicated. ChIP assay was carried out using ChIP grade anti-Sp1 antibody. The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 promoter bearing GC-box (Sp1 sites). Histogram shows the amplified DNA with real-time PCR analysis; open bars vs gray bars vs black bars. The data represent mean ± SD from two independent experiments (** p <0.05; * p <0.001). ( C ) The H 2 O 2 and/or UVB treatment schedule. ( D ) In vivo DNA binding assay revealed that transduction of Prdx6 and its mutant at K122/142R linked to TAT reactivated binding activity of Sp1 in aging primary hLECs. Primary hLECs of variable ages were transduced with TAT-HA-Prdx6 WT or its mutant TAT-HA-Prdx6K122/142R. ChIP experimentation was conducted using anti-Sp1 antibody. Immunoprecipitated DNA fragments were purified and processed for qPCR analysis using specific primers. Histograms represent the TAT-HA-Prdx6 WT and its mutant-induced enrichment of Sp1 at GC-box (Sp1 binding sites) in Prdx6 gene promoter. Open vs gray and black bars, and gray vs black bar; ** p <0.05, * p < 0.001. Data revealed a significant augmentation of Sp1 binding by TAT-HA-Prdx6K122/142R in all ages of LECs, but younger cells were more responsive.

Article Snippet: Sp1 activation assay was performed according to manufacturer’s protocol (TransAM Sp1 Transcription Factor Assay Kit, Cat No 41296, Active motif, Carlsland, California, USA).

Techniques: Binding Assay, Transduction, Mutagenesis, Recombinant, Amplification, Real-time Polymerase Chain Reaction, In Vivo, DNA Binding Assay, Activity Assay, Immunoprecipitation, Purification