Total proteins were isolated from BT20 cells using RIPA reagent (Invitrogen). The proteins were subject to separating in a 10% SDS/PAGE gel and then transferred onto a poly(vinylidene difluoride) (PVDF) membrane (Millipore). The PVDF membrane was incubated with a blocking buffer (3% BSA) for 1 h at room temperature and then incubated with anti‐KLF4 [Cell Signaling Technology (CST), Danvers, MA, USA; rabbit mAb #12173; 1 : 1000] or anti‐GAPDH (CST; rabbit mAb #5174; 1 : 1000) primary antibodies overnight at 4 °C. Then, the PVDF membrane was incubated with the secondary antibodies (Zhongshan Biotechnology, Beijing, China; goat anti‐rabbit; 1 : 10 000) for 1 h at room temperature. Finally, the PVDF membrane was exposed by using a SuperSignal West Dura Extended Duration Substrate Kit (Thermo Fisher Scientific, Beijing, China).
Anti klf4
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-KLF4 is a laboratory reagent used to detect and quantify the expression of the Kruppel-like factor 4 (KLF4) protein in biological samples. KLF4 is a transcription factor that plays a role in cellular differentiation and proliferation. The anti-KLF4 product can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to measure the levels of KLF4 in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti c myc, by Cell Signaling Technology (7 mentions)
Anti oct4, by Cell Signaling Technology (6 mentions)
Anti sox2, by Cell Signaling Technology (6 mentions)
Anti β actin, by Cell Signaling Technology (5 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti slug, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti nanog, by Cell Signaling Technology (2 mentions)
Anti sox2, by Santa Cruz Biotechnology (2 mentions)
Anti flag, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti oct4a, by Cell Signaling Technology (2 mentions)
Mg132, by Merck Group (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti survivin, by Cell Signaling Technology (2 mentions)
Anti muc1 c hm 1630 p1abx, by Thermo Fisher Scientific (2 mentions)
Anti n cadherin, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
32 protocols using anti klf4
1
Western Blot Analysis of KLF4 and GAPDH
2
Characterization of TNBC Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human TNBC cell lines MDAMB468, HCC1806, HCC1569, Hs578t, BT549, MDAMB231, and HCC1937 (Wu et al., 2015 (link)) were procured from American Type Culture Collection (ATCC, Manassas, VA), revived from early passage liquid nitrogen vapor stocks as required and maintained at 37°C in 5% CO2 and 95% humidity. All cells were authenticated via short tandem repeat testing. No mycoplasma contamination was noted. DAPT and GANT61 were procured from Sigma-Aldrich, St. Louis, MO, and Selleck Chemicals, Houston, TX. For Western blot, immunoprecipitation, immunofluorescence and immunohistochemistry, anti-ALDH1A, anti-Nanog, anti-Oct4, anti-KLF4, anti-c-MYC, anti-NICD, anti-JAGGED, and anti-Ki67 antibodies were purchased from Cell Signaling Technology, Beverly, MA. Antibodies anti-GLI1, anti-Sox2, anti-SHH, anti-FOXM1, and anti-HES1 were procured from Santa Cruz Biotechnology Inc Anti-HEY1 was purchased from ABclonal Technology, Woburn, MA. Mouse monoclonal β-Actin was procured from Sigma-Aldrich, St. Louis, MO. Horseradish peroxidase conjugated goat anti-rabbit IgG, goat anti-mouse IgG, and donkey anti-goat IgG were purchased from Sigma-Aldrich, St. Louis, MO. 3-(4,5-Dimethylthiazol-2-yl)–2,5-diphenyltetrazolium bromide (MTT) were procured from Sigma-Aldrich, St Louis, MO. Chemiluminescent peroxidase substrate was procured from GE Healthcare, UK.
3
Recombinant PRMT7 and Antibody Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-PRMT7 antibodies were purchased from Epicypher (#13-1009), Santa Cruz Biotechnology (SC9882) and Millipore (#07-639). Anti-Nanog (#61419), anti-Sox2 (#39823) and anti-H4R3me2s (#61187) antibodies were from Active Motif. Anti-Oct4 (#2840), anti-c-Myc (#5605) and anti-Klf4 (#4038) antibodies were from Cell Signaling. Anti-β-actin antibody (A5441) was from Sigma-Aldrich. Anti-H4R3me1 antibody (PA5-27065) was from Thermo Scientific. Anti-H3 antibody (ab1791) was from Abcam. Anti-mouse Argonaute 2 (Ago2) antibody was from Wako Chemicals (292-67301) and Cell Signaling (2897P). The cDNA constructs of human (OpenBiosystem) and mouse PRMT7 (OpenBiosystem) were cloned into pCDH1-EF1-IRES-GFP vector (System Biosciences) using the cloning sites EcoRI and NotI. Human PRMT7's catalytic mutant (m. PRMT7) was generated from pCDH1-EF1-PRMT7-IRES-GFP by mutating several residues (E144A, E153A, E478A and H644A) using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). These residues are likely located in arginine binding pockets according to crystal structures of PRMT1 and Caenorhabditis elegans PRMT7 (18 (link),19 (link)). Oligonucleotides used for cloning, ChIP-PCR, reverse-transcription (RT)-PCR, site-directed mutagenesis are listed in Supplementary Table S1.
4
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer supplemented with a protease inhibitor (Merck Millipore, Billerica, MA, USA). The protein lysates were quantified and subjected to SDS-PAGE, followed by electroblotting onto PVDF membrane. The membranes were incubated with anti-KLF4, -NANOG, -c-MYC or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (Cell Signaling Technology, Danvers, MA, USA). The reactive bands were visualized by chemiluminescence detection reagents (Merck Millipore).
5
Immunoblot Analysis of Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were isolated from cells transfected with the indicated vectors by using RIPA buffer containing protease inhibitor cocktail (P8340‐1ML, Sigma). A BCA protein assay kit (71285‐3, Millipore) was employed to measure the protein concentration. Immunoblot analysis was conducted with primary antibodies anti‐OTUD1 (ab122481, Abcam, 1:1000), anti‐Flag (F3165, MilliporeSigma, 1:1000), anti‐KLF4 (4038, Cell Signaling Technology, 1:1000), anti‐Myc (sc‐40, Santa Cruz Biotechnology, 1:1000) or anti‐ubiquitin (sc‐8017, Santa Cruz Biotechnology. 1:1000). After washing using phosphate buffer saline (PBS), the membranes were incubated with Rabbit anti‐Mouse IgG (H + L) secondary antibody, HRP (31 450, Invitrogen, 1:5000) or Goat anti‐Rabbit IgG (H + L) secondary antibody, HRP (31 460, Invitrogen, 1:5000). Finally, the signals of western blot analysis were observed using Immobilon ECL Ultra Western HRP Substrate (WBULS0100, Millipore).32
6
Stemness and Immune Markers Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Where indicated, cells were treated with RAR antagonist AGN193109 (5758, Tocris Bioscience, Bristol, UK) and RXR antagonist UVI3003 (3303, Tocris Bioscience) for 48 h (1 µM).
Total protein lysates were obtained using celLytic MT (C2978-50 mL, Sigma Aldrich), a cell lysis reagent, as described [32 (link)]. Antibodies used are as follows: anti-Oct-4A (2840 Cell Signaling, Milan, Italy), anti-Sox2 (3579 Cell Signaling), anti-KLF4 (4038 Cell Signaling), anti-Nanog (4903 Cell Signaling), anti-c-MYC (5605 Cell Signaling), anti-PD-L1 (13684; Cell Signaling); anti-ALDH3A1 (TA332730, Origene); anti-mPGES1 (160140) and anti-COX-2 (160112, Cayman Chemical, Arcore, Italy); anti-CD133 (PA1217, Boster), anti-NFkB (sc-372, Santa Cruz, CA, USA), and anti-β-actin (Sigma Aldrich). Images were digitalized with CHEMI DOC Quantity One program (Biorad, Milan, Italy
Total protein lysates were obtained using celLytic MT (C2978-50 mL, Sigma Aldrich), a cell lysis reagent, as described [32 (link)]. Antibodies used are as follows: anti-Oct-4A (2840 Cell Signaling, Milan, Italy), anti-Sox2 (3579 Cell Signaling), anti-KLF4 (4038 Cell Signaling), anti-Nanog (4903 Cell Signaling), anti-c-MYC (5605 Cell Signaling), anti-PD-L1 (13684; Cell Signaling); anti-ALDH3A1 (TA332730, Origene); anti-mPGES1 (160140) and anti-COX-2 (160112, Cayman Chemical, Arcore, Italy); anti-CD133 (PA1217, Boster), anti-NFkB (sc-372, Santa Cruz, CA, USA), and anti-β-actin (Sigma Aldrich). Images were digitalized with CHEMI DOC Quantity One program (Biorad, Milan, Italy
7
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction and western blotting methods were performed as described previously [53 (link)]. The antibodies used in this study including anti-β-actin (Santa cruz; sc-47778), anti-p38 (abcam; ab32142), anti-phopho-p38 MAPK(Cell signaling Technology; #9215), anti-SOX2 (Santa cruz; sc-20088), anti-Oct4 (abcam; ab19857), anti-HA (Cell signaling Technology, #3724), anti-c-Myc (Cell signaling Technology; #13987), anti-Nanog (abcam; ab80892), anti-Klf4 (Cell signaling Technology, #4038), anti-ERK (Beijing Zhongshan Golden Bridge Biotechnology Co Ltd, ZS94), anti-Ub (Santa cruz, sc-8017), anti-phospho-ATF2 (Cell signaling Technology, #9221). The anti-p38α, anti-p38β, anti-p38γ, anti-p38δ, anti-MKK3, anti-MKK6 antibodies were previously described by Kwong [26 (link)]. The p38 inhibitor SB203580 was from Sigma (s8307). For the protein stability assay of the stemness protein, cells were treated with cycloheximide (Cayman chemical, 14126) at 20 μg/ml concentration for 0h, 1h, 2h, 6h, 9h, 15h before lysates were collected. For the ubiquitylation assay, cells were treated with MG132 (Sigma, c2211) at 10 μM concentration for 6h before lysates were collected.
8
Immunohistochemical Analysis of KLF4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We acquired the tissue microarrays (TMAs) from Xinchao Biotech, Shanghai, China, comprising 46-paired tumor and non-tumor parts and 13 cases without corresponding non-tumor tissues. Antigen retrieval was performed after heating the slides in sodium citrate buffer (10 mM, pH6.0). The slides were incubated with anti-KLF4 (Cell Signaling Technology, USA) overnight at 4 °C after blocking with bovine serum albumin (Sango Biotech, China). Subsequently a secondary antibody was added to slides for incubation for 1 h at room temperature. A DAB solution was used for brown color development. Positivity rate of KLF4 was semi-quantified considering both the intensity and proportion of positive cells.
9
Protein Expression Analysis via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins were extracted from the treated cells using the RIPA lysis buffer method, and relatively quantified using BCA Protein Assay kit. The extracted proteins were loaded onto 10% SDS-polyacrylamide gels and electrophoresed fully. Subsequently, the separated proteins were transferred to a PVDF membrane using a wet transfer method. The membrane was blocked with 5% fat-free milk for 1 hour, and subsequently incubated with primary antibody (at 1:1000 ratio) at 4°C overnight. The membrane was washed 3 times (totally 30 min) with TBS-Tween buffer, and treated with secondary antibody (diluted with diluent as 1:5000 ratio) for 1 hour at room temperature. After washing 3 times with TBS-Tween buffer, the protein level was detected with enhanced chemi-luminescence (ECL) system (Pierce Biotechnology Inc., USA). The primary antibodies used are listed as follows: anti-GAPDH, anti-KLF4, anti-AKT, anti-p-AKT, anti-p21, anti-CCND1, anti-RB, anti-p-RB (Cell Signaling Technology, USA), anti-E-cadherin, anti-Fibronectin, anti-Snail, anti-Slug (Epitomics, USA).
10
Comprehensive Stem Cell Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-cIAP1 (#7065), Anti-cIAP2 (#3130), Anti-survivin (#2808) , Anti-Oct4 (#2750), Anti-klf4 (#12173), Anti-SUMO1 (#4930), Anti- HA-Tag (#3724), Phospho-p44/42 MAPK (Erk1/2) ( #4377), Cleaved-PARP (#5625) antibodies were purchased from Cell Signaling Technology; Anti-Naong ( ab21624), Anti-Sox2 ((ab92494) were obtained from Abcam; Anti-phospho-Sox2(S251) (AP3742a ) antibody was purchased from Abgent; Anti-LC3 (NB100-2220) antibody was obtained from Novus; Anti-Flag (F3165) and Anti-α-Tubulin (T9026) antibodies were obtained from Sigma; Anti-XIAP(sc-55551), Anti- ERK1/2(sc-292838), Anti-p62/SQSTM1 (sc-28359), Anti-caspase-3(sc-56046), goat anti-mouse IgG-HRP (sc-2005), goat anti-rabbit IgG-HRP (sc-2030) antibodies were purchased from Santa Cruz Biotechnology.
Bafilomycin A1, Chloroquine, MG-132, CHX were purchased from Sigma.
Bafilomycin A1, Chloroquine, MG-132, CHX were purchased from Sigma.
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!