Following the manufacturer’s protocol, ChIP Assay Kit (Beyotime) was applied for ChIP assay in transfected A172 and LN229 cells [28 (link)]. The cells were collected, immobilized in formaldehyde, and then lysed in ChIP lysis buffer. After the cross-linking of DNAs and proteins, the formaldehyde was quenched in glycine buffer. Then, sonication was used to generate DNA fragments (200–400 bp). Afterward, the fragmented DNAs were precipitated by anti-SP1 (Abcam). Anti-IgG (Abcam) was applied as the negative control. Finally, precipitated DNAs were determined by qPCR.
Anti sp1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-SP1 is a primary antibody that specifically binds to the transcription factor SP1. This antibody can be used in various applications to detect and study the expression of SP1.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Shanghai Enzyme-linked (3 mentions)
Normal rabbit igg, by Cell Signaling Technology (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti ubiquitin, by Abcam (2 mentions)
Anti p53, by Santa Cruz Biotechnology (2 mentions)
Anti ahr, by Cell Signaling Technology (2 mentions)
Anti vegfa, by Abcam (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Chip assay kit, by Beyotime (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Abcam (1 mentions)
Phalloidin 488, by Cytoskeleton (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Anti flag, by Proteintech (1 mentions)
Goat anti rabbit alexa 488, by Cell Signaling Technology (1 mentions)
Anti α syn antibody, by Santa Cruz Biotechnology (1 mentions)
Anti adh5, by Proteintech (1 mentions)
Goat anti mouse alexa 594, by Abcam (1 mentions)
33 protocols using anti sp1
1
ChIP Assay to Investigate SP1 Binding
2
Investigating ANGPTL7 and RhoA Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibodies were anti-ANGPTL7 (Abcam, Cambridge, UK), anti-RohA (CST, Beverly, MA, USA), anti-p-MLC (CST), anti-MLC (CST), anti-SP1 (Abcam), phalloidin-488 (Cytoskeleton, Denver, CO, USA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam). Y27632, a ROCK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA). Dexamethasone (Sigma D4902) was purchased from Sigma-Aldrich.
3
Antibody Characterization for Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies used in the present study were shown as follows: anti-α-SYN antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-MAOA antibody (Abcam, Cambridge, MA, USA), anti-Sp1 (Abcam, Cambridge, MA, USA), anti-R1 (ABclonal Technology, Wuhan, China), anti-Histone antibody (Cell Signaling Technology), anti-GAPDH antibody (Cell Signaling Technology, Danvers, MA, USA), anti-MAOB, anti-COMT, anti-ADH5, anti-FLAG, Goat anti-mouse IgG H&L and Goat anti-rabbit IgG H&L (Proteintech Group Inc., Rosemont, IL, USA), Goat anti-mouse Alexa 594 (Abcam) and Goat anti-rabbit Alexa 488 (Cell Signaling Technology).
4
Total Protein Extraction and Western Blot Analysis of Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extraction from cultured breast cancer cells were performed using RIPA lysis buffer containing 1X protease inhibitor cocktail. After protein concentrations measurement using the BCA method, equal protein (about 20–30 μg) from different samples were separated by 10% SDS‑PAGE and subsequently transferred into the polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA). Next, the membranes were immersed into 5% non-fat milk diluted in TBST for 1 h at room temperature, followed by incubation with the indicated primary antibodies overnight at 4 °C, including anti‑RSK4 (1:2000 dilution; No.ab76117, Abcam), anti‑SP1 (1:1000 dilution; No. ab227383, Abcam), anti-DNMT1 (1:2000 dilution; No. #5032, Cell Signaling Technology, MA, USA), anti-DNMT3A (1:2000 dilution; No. #2160, Cell Signaling Technology), anti-DNMT3B (1:2000 dilution; No. #67,259, Cell Signaling Technology), anti-Ub (1:1000 dilution; No. #3933, Cell Signaling Technology) and anti-GAPDH (No. ab181602, Abcam). After being washed with Tris Buffered Saline with Tween-20 (TBST) for 3 times, the membranes were probed with the corresponding secondary antibodies at room temperature for 1 h. Band intensity was measured using chemiluminescent reagents (Millipore) and quantified by ImageJ software (verson1.48, National Institutes of Health, MD, USA).
5
Immunoblotting Analysis of SP1 and MGMT in Glioma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA buffer (Cell Signaling Technology, U.S.A.) was used to homogenize the cells. The expression of SP1 and MGMT in glioma cells was detected by performing immunoblotting. Cells were lysed, cultured, or transfected in 1% PMSF supplemented RIPA buffer. Protein was loaded on to SDS/PAGE minigel, and then transferred on to PVDF membrane. The blots were probed with the following antibodies: anti-SP1 (Cat# EPR6662 (B), Abcam, U.S.A.), anti-MGMT (Cat# EPR4397, Abcam, U.S.A.), and anti-GAPDH (Cat# 6C5, Abcam, U.S.A.) at 4°C overnight, and incubated with HRP–conjugated secondary antibody (1:5000). Signals were visualized using ECL Substrates (Millipore, U.S.A.). The protein expression was normalized to endogenous GAPDH.
6
Chromatin Immunoprecipitation (ChIP) Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assays were performed essentially as described before22 ,26 (link),33 –44 . In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Santa Cruz, sc-10768), anti-Sp1 (Abcam, ab13370), anti-SRF (Cell Signaling Technology, 5147), (Santa Cruz, sc-585), anti-SLUG (Cell Signaling Technology, 9585), anti-ZEB1 (Cell Signaling Technology, 3396), anti-SNAIL (Cell Signaling Technology, 3879), anti-acetyl H3 (Millipore, 06-599), anti-acetyl H4 (Millipore, 06-598), anti-histone H3 (Millipore, 06-755), or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO3), diluted with the re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest.
7
Western Blot Analysis of Immune Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
THP‐1 or Raw264.7 cells were lysed with RIPA buffer mixed with protease and phosphatase inhibitors (100:1). Protein concentrations were quantified. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) were used to separate equal amounts of protein, which were then transferred to PVDF membranes (Millipore, Burlington, MA). After being blocked in 5% skimmed milk powder for 1 h at room temperature, the membranes were treated with the following primary antibodies overnight at 4°C: anti‐SP1 (Abcam), anti‐PSRC1 (GeneTex), anti‐ANXA2 (CST), anti‐p‐STAT3 (CST), anti‐STAT3 (CST), anti‐ERK1/2 antibody (Abcam), anti‐p‐ERK1/2 (Abcam), anti‐P38 (CST), anti‐p‐P38 (Proteintech) and anti‐β‐tubulin (Fude Biotech). The membranes were incubated wit secondary antibodies (Fude Biotech). Using an ECL kit, the protein bands were seen (Affinity, China) and analysed by ImageJ (National Institutes of Health, USA).
8
Protein Expression Analysis in Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from transfected cells using the RIPA lysis buffer (Shanghai Yeasen Biotechnology Co., Ltd.) followed by protein quantification using a BCA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.) and protein separation was performed on 10% gels using SDS-PAGE. PVDF membranes containing the proteins were blocked using 5% skimmed milk for 1 h at room temperature and incubated with anti-BST2 (1:1,000; cat no. ab243230), anti-SP1 (1:1,000; cat no. ab227383), anti-Ki67 (1:1,000; cat no. ab16667), anti-proliferating cell nuclear antigen (anti-PCNA; 1:1,000; cat no. ab18197), anti-MMP2 (1:1,000; cat no. ab92536) and anti-MMP9 (1:1,000; cat no. ab38898) primary antibodies (all from Abcam) at 4˚C overnight. Mouse anti-rabbit IgG HRP-conjugated secondary antibody (1:1,000; cat no. sc-2357; Santa Cruz Biotechnology, Inc.) was used for subsequent incubation with 2 h at room temperature. Protein bands were visualized using Immobilon™ Chemiluminescent HRP substrate (MilliporeSigma).
9
Protein Expression Analysis in A549 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein was extracted from A549 cells using RIPA buffer containing protease inhibitors and separated through Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Then, proteins were transferred to a PVDF membrane. After blocked with 5% fat‐free milk for 1 hour, the sample was incubated with primary antibodies for 1 hour and then with secondary antibodies for 1 hour. The following primary antibodies, namely anti‐Syncytin 1 (1:500), anti‐SP1 (1:1000) anti‐Active‐Caspase3 (1:1000), and anti‐Active‐Caspase9 (1:1000) were purchased from Abcam; anti‐Cyclin D1 (1:1000), anti‐Nusap1 (1:1000), anti‐CDK6 (1:1000), anti‐CDK4 (1:1000), anti‐Bcl2 (1:1000), anti‐Bax (1:1000), anti‐β‐catenin (1:1000), anti‐Vimentin (1:1000), anti‐E‐cadherin (1:1000), anti‐N‐cadherin (1:1000), anti‐P70 (1:1000), and anti‐GAPDH (1:5000) were purchased from Proteintech Group; anti‐p‐AKT (1:1000), anti‐p‐mTOR (1:1000), and anti‐p‐Erk1/2 (1:1000) were purchased from Cell Signaling Technology.
10
Protein expression analysis of TSA-treated cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 and THP-1 cells were treated with increasing concentrations of TSA (0, 1, 2.5, or 5 μM) or 0.1% DMSO (control). Western blot analysis was conducted as previously described [40 (link)]. The primary antibodies used were anti-GAPDH, anti-IRF5, and anti-Sp1 (Abcam) at dilutions of 1:200–3000.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!