Harvested FLS were directly lysed by RIPA buffer. Protein lysates were loaded onto SDS-PAGE gels, transferred to a PVDF membrane (Millipore, Darmstadt, Germany), and probed with an anti-KLF4 (1:500, Abcam) antibody, followed by incubation with peroxidase-conjugated Affinipure Goat Anti-Rabbit IgG (Jackson Immune Research, PA, USA). The horseradish peroxidase signal was generated using a western blot detection kit (Abfrontier, Seoul, Korea) and detected using a chemiluminescence system (GE, MA, USA).
Anti klf4
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-KLF4 is a primary antibody that specifically recognizes the Kruppel-like factor 4 (KLF4) protein. KLF4 is a transcription factor involved in the regulation of cell proliferation and differentiation. This antibody can be used for various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and study the KLF4 protein.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Anti sox2, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti nanog, by Abcam (2 mentions)
Anti muc2, by Abcam (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti sox2, by Cell Signaling Technology (2 mentions)
Chemiluminescence system, by GE Healthcare (1 mentions)
Western blot detection kit, by AbFrontier (1 mentions)
Microson ultrasonic cell disruptor xl, by Bioventus (1 mentions)
Simplechip plus sonication chromatin ip kit, by Cell Signaling Technology (1 mentions)
Anti h4k20me1, by Abcam (1 mentions)
Anti oct4, by Abcam (1 mentions)
Anti nanog, by Active Motif (1 mentions)
Anti ctcf, by Merck Group (1 mentions)
Anti brd4, by Fortis Life Sciences (1 mentions)
Anti cebpb, by Abcam (1 mentions)
Anti ets1, by Active Motif (1 mentions)
Anti ikaros, by Active Motif (1 mentions)
22 protocols using anti klf4
1
Western Blot Analysis of KLF4 Protein Expression
2
ChIP-qPCR Analysis of KLF4 and H4K20me1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation (ChIP) assays were conducted with Simple ChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology, MA) according to the manufacturer’s instructions. Briefly, cells (1 × 107) were fixed with 1% formaldehyde for 10 min at room temperature to cross-link DNA and proteins. Glycine was then added to stop the cross-linking. Chromatin was sheared using Microson Ultrasonic Cell Disruptor XL (Misonix) with 16 cycles of sonication (15 s each, 2-min rest; amplitude = 10, power = 15 W). Ten-microliter sonication solution was taken out from each sample as the input control, and the remaining was incubated with anti-KLF4 (Abcam, USA) or anti-H4K20me1 (Abcam, USA), or histone H3-positive control, or IgG negative control at 4 °C overnight. Immunoprecipitates were bound to protein G magnetic beads, and the DNA–protein cross-link was reversed at 65 °C for 2 h. DNA was purified and enrichment of DNA sequences was detected using qPCR. SIRT4 oligonucleotide sequences for PCR primers were forward 5′-GAAGAGATGGGATCTCACTTTGTC-3′ and reverse 5′-GTAGACAACCAGAACTGCCGCTCT-3′.
3
Histone Modifications and Chromatin Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit polyclonal to H1, HMGN1, HMGN2, and H3 were from our laboratory, anti H3K27ac (Abcam#ab4729), anti H3K27me3 (Abcam#ab6002), monoclonal anti H1(Milipore-Sigma #05-457), anti CEBPB (Abcam#ab32358), Anti-Brd3 (Active Motif #61489), Anti-Brd4 (Bethyl Laboratories #A301-985A100), Anti-CEBPB (Abcam #ab32358), Anti-CTCF (EMD Millipore #07-729), Anti-Ets1 (Active Motif #39580), Anti-Ikaros (Active Motif #39355), Anti-Irf8 (Bethyl Laboratories #A304-027A), Anti-Klf4 (Abcam #106629), Anti-Nanog (Active Motif #61419), Anti-Oct4 (Abcam #ab19857), Anti-p300 (Active Motif #61401), Anti-Pax5 (Abcam #183575), Anti-Sox2 (Abcam #97959).
The following recombinant mononucleosomes were purchased from Active Motif: unmodified (#81070); H3K27me3 modified (#81834), H3K27ac modified (#81077).
Wild type and HMGN DKO mouse embryonic fibroblasts, embryonic stem cell lines55 (link) and resting B cells56 (link) were as previously described. Peptides Histone H3 (23–34) peptide, KAARKSAPATGG and Histone H3K27ac (23–34) peptide, KAAR - K(Ac)—SAPATGG were from AnaSpec, Inc.
The following recombinant mononucleosomes were purchased from Active Motif: unmodified (#81070); H3K27me3 modified (#81834), H3K27ac modified (#81077).
Wild type and HMGN DKO mouse embryonic fibroblasts, embryonic stem cell lines55 (link) and resting B cells56 (link) were as previously described. Peptides Histone H3 (23–34) peptide, KAARKSAPATGG and Histone H3K27ac (23–34) peptide, KAAR - K(Ac)—SAPATGG were from AnaSpec, Inc.
4
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was isolated from cell lysates or tissues by using RIPA buffer and then quantified by BCA protein assay kit (Beyotime, Shanghai, China). Proteins were resolved on 10% SDS-PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. The primary antibodies were as follows: anti-p38 (Abcam, Cambridge, MA, USA; 1:1000), anti-ERK (Abcam; 1:1000), anti-JNK (Abcam; 1:1000), anti-KLF4 (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). The density of the bands was measured using ImageJ software. Signals were detected with enhanced chemiluminescence using β-actin as the internal standard (Kodak).
5
Western Blot Analysis of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested, washed with PBS, and lysed with lysis buffer supplemented with protease inhibitors (Roche, Sainte‐Agathe‐Nord, QC, Canada). After the protein concentrations were determined using a Bio‐Rad DC protein assay kit (Bio‐Rad, Hercules, CA, USA), samples were then normalized and denatured. The samples were then loaded into an 8% polyacrylamide gel and separated by SDS/PAGE followed by transference to a PVDF membrane. Proteins were identified by incubation with primary antibodies followed by horseradish peroxidase‐conjugated secondary antibodies and an enhanced chemiluminescence solution (Thermo Scientific, Waltham, MA, USA). Antibodies used in this study include the following: anti‐YAP1(1 : 1000, Cat: 4912; Cell Signaling, Cambridge, MA, USA), anti‐CD44 (8E2) monoclonal antibody (1 : 1000, Cat: 5640; Cell Signaling), anti‐ALDH1A1 (1 : 1000, Cat: ab105920; Abcam, Toronto, ON, Canada), anti‐Klf4 (1 : 1000, Cat: ab72543; Abcam), anti‐β‐catenin (1 : 1000, Cat: 610153, Clone 14; BD, Mississauga, ON, Canada), anti‐active β‐catenin (1 : 500, Cat: 05665, Clone 8E7; Millipore, Billerica, MA, USA), and anti‐α‐tubulin monoclonal antibody (1 : 500, Cat: T9026; Sigma‐Aldrich).
6
Antibody Characterization in Cell Studies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for western blot analysis and
immunofluorescence staining: anti-BCL2 (1:500, Abcam, Cambridge, MA,
USA), anti-KLF4(1:500, Abcam), anti-SOX2 (1:500, Abcam), anti-NANOG
(1:200, Abcam), anti-CASPASE3 (1:1000, Cell Signaling Technology,
Danvers, MA, USA), anti-MMP2 (1:1000, Cell Signaling Technology),
anti-MMP14 (1:1000, Cell Signaling Technology), anti-Laminin (1:1000,
Abcam), anti-involucrin (1:500, Abcam), anti-col Ⅰ (1:1000, Abcam),
anti-beta actin (1:5000, Sigma-Aldrich, St. Louis, MO, USA), goat
anti-mouse IgG-HRP (1:5000), and rabbit IgG-HRP (1:10,000, Bethyl
Laboratories, Montgomery, TX, USA).
immunofluorescence staining: anti-BCL2 (1:500, Abcam, Cambridge, MA,
USA), anti-KLF4(1:500, Abcam), anti-SOX2 (1:500, Abcam), anti-NANOG
(1:200, Abcam), anti-CASPASE3 (1:1000, Cell Signaling Technology,
Danvers, MA, USA), anti-MMP2 (1:1000, Cell Signaling Technology),
anti-MMP14 (1:1000, Cell Signaling Technology), anti-Laminin (1:1000,
Abcam), anti-involucrin (1:500, Abcam), anti-col Ⅰ (1:1000, Abcam),
anti-beta actin (1:5000, Sigma-Aldrich, St. Louis, MO, USA), goat
anti-mouse IgG-HRP (1:5000), and rabbit IgG-HRP (1:10,000, Bethyl
Laboratories, Montgomery, TX, USA).
7
Western Blotting Protocol for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed according to standard procedures. We used the following primary antibodies: anti-HOOK1 (Abcam, ab151756), anti-CASP3 (Cell Signaling, #9664), anti-CASP9 (Cell Signaling, #9502), anti-PARP (Cell Signaling, #9532), anti-p-H2AX (Ser139) (Cell Signaling, #9718), anti-NANOG (Santa Cruz Biotechnology, sc-293,121), anti-OCT3/4 (Santa Cruz Biotechnology, sc-5279), anti-KLF4 (Abcam, ab72543), anti-ATF6α (Santa Cruz Biotechnology, sc-166,659), anti-ATF4 (Santa Cruz Biotechnology, sc-390,063), anti-GRP78 (Santa Cruz Biotechnology, sc-13,539), anti-CHOP (Cell Signaling, #2895), anti-LC3B (Abcam, ab48394), anti-p62 (Abcam, ab109012), and anti-B-actin (Abcam, ab16039) as a loading control. We used the following secondary antibodies: rabbit anti-mouse (Abcam, ab97046) and goat anti-rabbit (Abcam, ab97051). The proteins were detected using an ECL detection system (Amersham Biosciences) and a Bio-Rad Chemidoc Touch.
8
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the proteins were extracted using RIPA buffer containing PMSF (Beyotime, China). The protein samples were loaded onto the SDS-PAGE gels and electrotransferred to PVDF membranes (Milipore, America). The membranes were blocked with 5% nonfat milk for 2 hours, and incubated overnight at 4°C with specific primary antibodies, i.e., anti-hTERT (1:1000, Abcam, USA), anti-KLF4 (1:1000, Abcam, USA), anti-p65 (1:1000, Abcam, USA), anti-p50 (1:1000, Abcam, USA), anti- CDX2 (1:1000, Abcam, USA), anti-MUC2 (1:1000, Abcam, USA) and anti-GAPDH (1:500, Boaoshen, China) antibodies. The membranes were subsequently incubated with a secondary antibody. The bands were quantified using Quantity One software (Bio-Rad, USA), and the gray value was analyzed using GraphPad Prism software (GraphPad, USA).
9
Protein Expression Analysis in A549 and H520 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 and H520 cells were harvested and lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). The BCA assay kit (Bio-Rad Laboratories, Inc.) was used to determine protein concentration. The protein samples (40 μg per lane) were loaded and electrophoresed via SDS-PAGE on a 10% gel, and then transferred to PVDF membranes (EMD Millipore). Membranes were blocked with 5% skimmed milk at room temperature for 1 h, and subsequently co-incubated with the primary antibodies at 4˚C overnight. Subsequently, the bound primary antibodies were incubated with the corresponding secondary antibodies (1:5000, cat. no. ab6721, Abcam) overnight at 4˚C, and then detected by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). The primary antibodies used were purchased from Abcam and were as follows: Anti-KLF4 (1:1000; cat. no. ab215036; Abcam), anti-MSI2 (1:2000; cat. no. ab76148; Abcam), anti-JAK2 (1:1000; cat. no. ab108596; Abcam), anti-phosphorylated (p)-JAK2 (1:1000; cat. no. ab32101; Abcam), anti-STAT3 (1:2000; cat. no. ab68153; Abcam), anti-p-STAT3 (1:1000; cat. no. ab76315; Abcam), anti-Wiskott-Aldrich syndrome protein family member 3 (WASF3; 1:2000; cat. no. ab68031; Abcam) and anti-GAPDH (1:1,000; cat. no. ab9484; Abcam). ImageJ software (version 2.0; National Institutes of Health) was used for semi-quantification of the band densities.
10
Western Blot Analysis of Mechanically Stretched AD-VSMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer (strong) RIPA (YEASEN, 20101ES60, USA) was used to extract proteins from mechanically stretched and unstretched AD‐VSMCs. The protein concentration was determined using the BCA method, and Western blotting was then used to detect protein levels. Briefly, protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were then blocked with 5% skim milk for 2 h and incubated with primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with secondary antibodies for 1 h. Visualization was performed using an enhanced chemiluminescence detection system, and Photoshopcs3 software was used to analyze the relative band intensity. The primary antibodies used in this study were as follows: anti‐α‐SMA antibodies (Abcam, Cambridge, UK; cat. no. ab7817), anti‐SM22‐α antibodies (Abcam; cat. no. ab14106), anti‐OPN antibodies (Abcam; cat. no. ab8448), anti‐PCNA antibodies (Abcam; cat. no. ab29), anti‐KLF4 (Abcam; cat. no. 106629), anti‐MMP9 antibody (Abcam; cat. no. ab76003), anti‐GAPDH antibody (Abcam; cat. no. ab9485) and anti‐β‐actin antibody (Arigo; cat. no. arg62346).
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!