sp1 d4c3 rabbit mab  (Cell Signaling Technology Inc)

Journal: Molecular cell

Article Title: Tandem deubiquitination and acetylation of SPRTN promotes DNA-protein crosslinks repair and protects against aging

doi: 10.1016/j.molcel.2020.06.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-SP1 (D4C3) , Cell Signaling Technology , Cat# 9389; RRID:AB_11220235.

Techniques: Recombinant, Staining, Mutagenesis, In Situ, Fractionation, Software

Journal: PLoS ONE

Article Title: Effect of differentiation, de novo innervation, and electrical pulse stimulation on mRNA and protein expression of Na + ,K + -ATPase, FXYD1, and FXYD5 in cultured human skeletal muscle cells

doi: 10.1371/journal.pone.0247377

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: Sp1 (D4C3) , Rabbit , Monoclonal , 1:1000 , CST , 9389.

Techniques:

Journal: PLoS ONE

Article Title: Effect of differentiation, de novo innervation, and electrical pulse stimulation on mRNA and protein expression of Na + ,K + -ATPase, FXYD1, and FXYD5 in cultured human skeletal muscle cells

doi: 10.1371/journal.pone.0247377

Figure Lengend Snippet: (A) Human skeletal muscle cells were isolated from samples of m . semitendinosus and grown in Advanced MEM with 10% FBS. Before reaching confluence, cells were separated into CD56 + and CD56 - fractions using MACS microbeads. CD56 + and CD56 - cells were subsequently grown in Advanced MEM with 10% FBS (CD56 + 10% FBS and CD56 - 10% FBS cells) or were differentiated for 7 days in Advanced MEM with 2% FBS (CD56 - 2% FBS and CD56 + 2% FBS cells). (B-I) qPCR was used to estimate gene expression of (B) NCAM1 (CD56), (C) desmin, (D) myosin heavy chains MYH1 , MYH2 , and MYH7 , (E) CACNA1S , (F) SERCA1 ( ATP2A1 ) and SERCA2 ( ATP2A2 ), (G) NKAα1–3 ( ATP1A1-3 ), (H) NKAβ1–3 ( ATP1B1-3 ), and (I) FXYD1 and FXYD 5. A geometric mean of 18S rRNA and cyclophilin (PPIA) was used for normalization of gene expression. (J-N) Immunoblotting was used to estimate protein abundance of (J) myosin heavy chains MyHC-2A and -2X, and MyHC-β/slow, (K) NKAα1 and NKAα2, (L) FXYD1 and FXYD5, (M) Sp1, and (N) actin. Note that in humans, SC-71 antibody detects MyHC-2A and -2X (see ). Results are means ± SEM (n = 3–4), * P <0.05 vs. 10% FBS, # P <0.05 vs CD56.

Article Snippet: Sp1 (D4C3) , Rabbit , Monoclonal , 1:1000 , CST , 9389.

Techniques: Isolation, Gene Expression, Western Blot, Quantitative Proteomics

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma

doi: 10.3390/ijms22041716

Figure Lengend Snippet: Effect of tumor necrosis factor-alpha (TNF-α) and MLN4924 on matrix metalloproteinase 9 (MMP9) expression and esophageal squamous cell carcinoma (ESCC) cell migration. ( A ) After overnight serum starvation, KYSE150 cells were pretreated or not with 1 μM MLN4924 for 30 min and then stimulated with TNF-α at the indicated concentration for 24 h. Activity of MMP9 and MMP2 was analyzed by gelatin zymography in the conditioned media. The protein levels of membrane type I-matrix metalloproteinase (MT1-MMP), cyclin dependent kinase inhibitor 1A (CDKN1A/p21), c-Jun, nuclear factor kappa B (NFκB) and SP1 were determined by Western blotting. Fold change calculated as the ratio of relative levels of proteins normalized to β-actin (ACTB) between the cells treated with TNF-α in combination with MLN4924 and the cells treated with TNF-α alone is shown in . Values shown are means ± SEM: whiskers: min–max. ( B ) MMP9 messenger RNA (mRNA) expression was analyzed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) in both KYSE150 and KYSE70 cells treated for 24 h with TNF-α (30 ng/mL) and MLN4924 (1 µM) alone or in combination. Results are presented as fold change of MMP9 gene in the treated cells relative to the untreated controls normalized to the expression of the reference gene encoding glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). ( C ) Representative images from wound healing assay showing the changes in KYSE70 cell migration under MLN4924 and TNF-α applied separately and in combination after 24 h and 48 h treatment in relation to the untreated controls. The cells were seeded in silicone inserts and allowed to attach and form a confluent monolayer. After the inserts were removed, the cells were treated with TNF-α (30 ng/mL) and MLN4924 (1 µM) separately or in combination and incubated for the next 48 h. Images of cell-free artificial wounds were taken at 0, 24 and 48 h. ( D ) Quantitative analysis of wound closure. Summary bar graphs illustrating the ratio of wound closure after 24 and 48 h quantified for KYSE150 and KYSE70 cells. Values shown are means ± SEM. * p < 0.001, ** p < 0.05 vs. control. Ctrl—untreated controls.

Article Snippet: Rabbit anti-β-Actin (13E5) (4970), c-Jun (60A8) (9165), Phospho-c-Jun (Ser73) (D47G9) XP ® (3270), Phospho-IκBα (Ser32) (14D4) (2859), Phospho-NF-κB p65 (Ser536) (3033), p21 Waf1/Cip1 (12D1) (2947), SP1 (D4C3) (9389S), mouse anti-IκBα (L35A5) (4814) and NF-κB p65 (L8F6) (6956) antibodies were products of Cell Signaling.

Techniques: Expressing, Migration, Concentration Assay, Activity Assay, Zymography, Membrane, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Wound Healing Assay, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma

doi: 10.3390/ijms22041716

Figure Lengend Snippet: Western blot analysis of the nuclear factor kappa B (NFκB) complex and the chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) assay of the matrix metalloproteinase 9 ( MMP9 ) gene promoter in KYSE150 cells. Nuclear protein-enriched extracts from esophageal squamous cell carcinoma (ESCC) cells were used for co-immunoprecipitation (co-IP) with a mouse anti-NFκB/p65 antibody ( A ) or with a rabbit anti-c-Jun antibody ( B ) and probed for inhibitor of nuclear factor kappa B-alpha (IκB-α)/phospho-IκB-α, SP1 and cyclin dependent kinase inhibitor 1A (CDKN1A/p21) as well as phosphorylated and unphosphorylated forms of NFκB/p65 and c-Jun. Immunoglobulin G (IgG) mixture (1:1:1) of each protein extract and normal mouse IgG or normal rabbit IgG, respectively, used as negative controls; Ctrl—protein extract from untreated cells; MLN4924 and tumor necrosis factor-alpha (TNF-α)—extracts from cells treated with MLN4924 (1 µM) for 24 h and with TNF-α (30 ng/mL) for 1 h, respectively. ( C ) ChIP-qPCR assay on the MMP9 gene promoter in KYSE150 cells. Sheared chromatin was immunoprecipitated with antibodies against NFκB/p65 and with an anti-acetyl histone H3.3 antibody, used as a positive control. Normal rabbit IgG served as a negative antibody control. Box plots showing qPCR data normalized with the fold enrichment method. Values shown are means ± SEM: whiskers: min–max; * p < 0.001, ** p < 0.01 vs. controls. Agarose gels showing DNA fragments amplified with MMP9 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers.

Article Snippet: Rabbit anti-β-Actin (13E5) (4970), c-Jun (60A8) (9165), Phospho-c-Jun (Ser73) (D47G9) XP ® (3270), Phospho-IκBα (Ser32) (14D4) (2859), Phospho-NF-κB p65 (Ser536) (3033), p21 Waf1/Cip1 (12D1) (2947), SP1 (D4C3) (9389S), mouse anti-IκBα (L35A5) (4814) and NF-κB p65 (L8F6) (6956) antibodies were products of Cell Signaling.

Techniques: Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Co-Immunoprecipitation Assay, ChIP-qPCR, Positive Control, Control, Amplification

Journal: Cell Death & Disease

Article Title: Targeting the NCOA3-SP1-TERT axis for tumor growth in hepatocellular carcinoma

doi: 10.1038/s41419-020-03218-x

Figure Lengend Snippet: A Illustration of the SP1 binding sites in the TERT promoter. B The interaction between NCOA3 and SP1 was tested by co-IP in Hep3B and HepG2 cells without (vector) or with NCOA3 overexpression (N3). NCOA3 antibody was added to the nuclear extracts to precipitate proteins interacting with NCOA3, while IgG was used as a negative control. SP1 was then detected by its specific antibody in the precipitates. C The 5′ biotin-labeled TERT promoter probe was pulled down by NCOA3 antibody in immortalized LO2 and HCC (Hep3B, HepG2, BEL-7402, and SNU-449) cells, while a nonspecific probe (NSP) was used as a negative control. SP1 was detected by its specific antibody in the precipitates. D Immunofluorescence of NCOA3 (red) and SP1 (green) in Hep3B and HepG2 cells. E TERT expression was detected in Hep3B cells without (Sp1-RNAi-NC) or with SP1 knockdown (Sp1-RNAi-1, Sp1-RNAi-2). F Viability of Hep3B cells with NCOA3 overexpression and SP1 knockdown. The data were shown as means ± SD, * P < 0.05, ** P < 0.01, Student’s t test from three independent experiments.

Article Snippet: Anti-NCOA3 antibody (#2126), anti-SP1 (D4C3) antibody (#9389), and anti-Acetylated-Lysine antibody (#9441) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Over Expression, Negative Control, Labeling, Immunofluorescence, Expressing, Knockdown