Samples were resolved on 8% SDS-PAGE, transferred onto nitrocellulose membranes (Bio-Rad), and incubated with primary antibodies against STEP (1:1000, Millipore), non-phospho-STEP (1:1000, Cell Signaling), phospho-Tyr (1:2000, Millipore), GluN2A (1:2000, Millipore), GluN2B (1:2000, Millipore), pY416 Src (1:1000, Cell Signaling), Src (1:2000 Santa Cruz), Fyn (1:2000 Santa Cruz), and β-actin (1:5000, Santa Cruz) overnight at 4 °C, and HRP-conjugated secondary antibodies (1:5000; Pierce) or Clean-Blot IP detection reagent HRP (1:1000; Thermo Scientific, Rockford, IL) for 2 h at RT. Immunoreactivity was developed with a Chemiluminescent Substrate kit (Pierce) and visualized by G:BOX with the GeneSnap software (Syngene, Cambridge, UK). All densitometric quantification was obtained using ImageJ software (National Institutes of Health).
Clean blot ip detection reagent hrp
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Clean-Blot IP Detection Reagent (HRP) is a laboratory reagent used for the detection of proteins in immunoprecipitation (IP) experiments. It is a horseradish peroxidase (HRP)-conjugated secondary antibody that binds to the primary antibody used in the IP process, allowing for the visualization of the target protein.
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Lab products found in correlation
Molecular imager chemidoc xrs system, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Bp 110r, by Boston BioProducts (2 mentions)
Src py416, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Glun2a, by Merck Group (1 mentions)
Glun2b, by Merck Group (1 mentions)
Genesnap software, by Syngene (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg h l hrp, by GenDEPOT (1 mentions)
Goat anti mouse igg h l hrp, by GenDEPOT (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Anti rabbit igg antibody, by GE Healthcare (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Pirarubicin, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse igg, by GE Healthcare (1 mentions)
Daunorubicin, by Merck Group (1 mentions)
Idarubicin, by Merck Group (1 mentions)
Recombinant human tnf α, by R&D Systems (1 mentions)
1640 media, by Thermo Fisher Scientific (1 mentions)
15 protocols using clean blot ip detection reagent hrp
1
Quantifying STEP and Glutamate Receptor Signaling
2
Denaturing Protein Analysis via Western Blot
3
Endogenous and Exogenous Protein Interactions
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Endogenous immunoprecipitation was performed using PureProteome Protein A/G Magnetic Bead System (LSKMAGA10) according to the manufacturer's protocol with slight modification. Briefly, cells were lysed in ice cold RIPA ((50 × 10−3 m Tris (pH 7.3), 150 × 10−3 m sodium chloride, 0.25 × 10−3 m EDTA, 1% Triton X‐100, 1% w/v sodium deoxycholate, and a mixture of protease inhibitors) and split equally to be precleared with IgG control or BNIP‐2 antibody for 45 min at 4 °C. Protein A magnetic beads were then added and the sample was further incubated for 2 h with constant shaking at 4 °C. Samples were washed thrice with ice cold 1× PBS before adding loading dye and denatured at 95 °C for 10 min.
For exogenous co‐immunoprecipitation, anti‐Flag‐tagged or anti‐HA‐tagged magnetic beads from Sigma‐Aldrich were incubated with cell lysates lysed in ice cold RIPA for 1 h at 4 °C prior to washing thrice with 1X PBS. Immunoprecipitation for the active (GTP‐bound) form of RhoA was performed as described previously.[44 ] Western blot was performed as previously described.[22 ] Blots were analyzed using Bio‐Rad clarity ECL substrate (1:3000 dilution) in a ChemiDoc Touch (Bio‐Rad) developer. For endogenous IP, Clean‐Blot IP Detection Reagent (HRP) (#21230) from ThermoFisher Scientific was used at 1:500 dilution.
For exogenous co‐immunoprecipitation, anti‐Flag‐tagged or anti‐HA‐tagged magnetic beads from Sigma‐Aldrich were incubated with cell lysates lysed in ice cold RIPA for 1 h at 4 °C prior to washing thrice with 1X PBS. Immunoprecipitation for the active (GTP‐bound) form of RhoA was performed as described previously.[
4
Anticancer Agents and Molecular Targets
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Ten milligrams of epirubicin hydrochloride injection “NK” was purchased from Nippon Kayaku and dissolved in normal saline (Otsuka) at the time of use for in vitro and in vivo experiments. Pirarubicin, doxorubicin, daunorubicin and idarubicin were all purchased as hydrochloride salts from Sigma-Aldrich. Recombinant human TNF-α was purchased from R&D Systems. Anti-Foxp3 and anti-GAPDH antibodies were purchased from Abcam. Anti-NFAT1 and anti-NF-κB antibodies were purchased from Cell Signaling Technologies. Anti-Foxp3 antibody for immunoprecipitation was purchased from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG antibodies were purchased from GE Healthcare. Clean-Blot™ IP Detection Reagent (HRP) was purchased from Thermo Scientific.
5
Cell Culture and Protein Analysis
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A549 and H1299 cells were cultured in 1640 media (GIBCO) supplemented with 10% FBS at 37 °C in 5% CO2 humidified incubators. MG132 (cat. C2211) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Thymidine (cat. 6060) was acquired from Millipore (Burlington, MA, USA). GeneMut siRNA transfection reagent (cat. SL100568) was purchased from SignaGen Laboratories (Rockville, MD, USA). LipoMax plasmid transfection reagent (cat. 17052012) was purchased from SUDGEN (Bellevue, WA, USA). M-PER lysis buffer (cat. 78501), Dynabeads (cat. 10004D) and Clean-blot™ IP detection reagent (HRP) (cat. 21230) were purchased from Thermo Fisher (Waltham, MA, USA). RIPA buffer (cat. P0013B) and Cell cycle and apoptosis analysis kit (cat. C1052) were purchased from Beyotime (Shanghai, China). The protease inhibitor cocktail (cat. B14012) and Cell Counting Kit-8 solution (CCK-8, cat. B34304) were purchased from Bimake (Houston, TX, USA). HRP-labeled goat anti-mouse (cat. GB23301) and goat anti-rabbit secondary antibody (cat. purGB23303) were purchased from Servicebio (Wuhan, China).
6
Protein Extraction and Western Blotting
7
Embryonic Ectodermal Protein Enrichment
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E13.5 CM was collected by manual dissection and the CM was enriched from the ectoderm (described above). At least 2 million cells from multiple embryos were incubated with 1 ml lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 2 mM EGTA, and 1% Triton X-100 in H2O) and disrupted with a 23-gauge needle and syringe. The precipitating antibody was added to the lysate and incubated overnight at 4°. Immunoprecipitation was performed by incubating 100 µl of Dynabeads Protein G beads (Invitrogen 10003D) with the lysate and antibody for 1 hr at 4°. Beads were then washed four times with 1 ml wash buffer (40 mM HEPES, 300 mM NaCl, 10% Glycerol, and 0.2% NP40 in H2O). The sample was collected in NuPage LDS Sample Buffer (Thermo Fisher Scientific NP0007) supplemented with β-mercaptoethanol, and was heated at 90° for 5 min. Rabbit polyclonal antibodies against nonphospho-β-catenin, 3.43 µg (Cell Signaling D13A1 #8814); EZH2, 10 µg (Diagenode C15410039); and IgG, 6 µg (Abcam ab46540) were used for immunoprecipitation. Protein species were separated by SDS-PAGE using Mini-PROTEAN TGC gels (Bio-Rad #456-1084). Western blots were performed with primary antibodies against β-catenin (1:1000; Millipore 06-734) or EZH2 (1:500, Cell Signaling #5246). Clean-Blot IP Detection Reagent (HRP) (1:250; Thermo Fisher Scientific 21232) was used as secondary antibody.
8
Phospho-specific Immunoprecipitation and Detection
9
Co-immunoprecipitation of MCL-1 and DRP-1
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CAFs were collected in lysis buffer (CHAPS 1%, TRIS-HCl 20 mM pH=7.5, NaCl 150 mM, EDTA 1 mM pH = 8.0, proteases and phosphatases inhibitors). Co-Immunoprecipitation was performed using PureProteome™ Protein A Magnetic Beads (Millipore, LSKMAGA10). Protein extracts were first precleared using 10 µl of beads for 200 µg of proteins (1-h incubation at 4 °C). 2 µl of anti-MCL-1 antibody (Cell signaling mAb #94296) and 10 µl of beads were then used for 200 µg of proteins following manufacturer’s instructions. For western blotting, DRP-1 antibody (MA5-26255, Thermo Fisher Scientific, Waltham, MA, USA) was used as primary antibody (1:1000) and the same anti-MCL-1 primary antibody (1:1000). Clean-Blot™ IP Detection Reagent (HRP) (Thermo Scientific #21230) was used as secondary antibody (1:1000). Whole uncropped images of the original western blots are presented in Supplementary Fig. 4 .
10
Detailed Immunoblotting and Immunofluorescence Protocol
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Clean-Blot IP detection reagent (HRP) (21230) for clean IP detection was purchased from Thermo Scientific Company. The primers sequences for WTX gene CDS and qRT-PCR were listed in Supplementary Tables 2 and 3 . GeneChem (ShangHai, China) synthesized the siRNAs, the sequences shown in Supplementary Table 4 . Primary antibodies information shows in Supplementary Table 7 . Secondary antibodies conjugated to Alexa 488 or Alexa 594 for immunofluorescence staining and conjugated to HRP for Immunoblotting were purchased from ZSGB-BIO (Guangzhou, China), Rhodamine Phalloidin was purchased from Cytoskeleton Inc (USA). Tissue samples were obtained from patients who had undergone routine surgery at Nanfang Hospital, Southern Medical University, China, between 2005 and 2015. The informed consent was obtained from all human participants.
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