Western blotting was performed as previously described according to the standard protocol [57 ,58 (link)]. The primary antibodies and concentrations used for western blotting were: PARP1 (Cell Signaling Technology, 1:1000 dilution), SLC7A11/xCT (D2M7A) (Cell Signaling Technology, 1:1000 dilution), p53 (Cell Signaling Technology, 1:1000), GPX4 (Abcam-ab125066, 1:1000 dilution), BAP1 (Cell Signaling Technology, 1:1000), ATF3 (Cell Signaling Technology, 1:1000), NRF2 (Cell Signaling Technology, 1:1000), Vinculin (Cell Signaling Technology, 1:3000), Tubulin (Cell Signaling Technology, 1:3000), phospho-histone H2A.X (Ser139) (Cell Signaling Technology, 1:1000), phospho-Chk2 (Thr68) (Cell Signaling Technology, 1:1000), phospho-ATM (Ser1981) (Cell Signaling Technology, 1:1000), cleaved Caspase-3(Cell Signaling Technology, 1:1000). The secondary antibodies used were: horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology, 1:5000 dilution), horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling Technology, 1:5000 dilution). Proteins were visualized with the ECL Western blotting substrate (32,109, ThermoScientific, USA).
Horseradish peroxidase conjugated anti mouse igg
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is conjugated with the enzyme horseradish peroxidase. This product is used to detect and visualize mouse primary antibodies in various immunoassay techniques, such as Western blotting and enzyme-linked immunosorbent assay (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000, by GE Healthcare (3 mentions)
Las 4000, by Cytiva (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti rabbit igg secondary antibody, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Phospho histone h2a x ser139, by Cell Signaling Technology (1 mentions)
Phospho atm ser1981, by Cell Signaling Technology (1 mentions)
Phospho chk2 thr68, by Cell Signaling Technology (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
A2228, by Merck Group (1 mentions)
Anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Ecl plus kit, by Thermo Fisher Scientific (1 mentions)
23 protocols using horseradish peroxidase conjugated anti mouse igg
1
Comprehensive Western Blot Analysis
2
Western Blot Analysis of PPARγ
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The 5637 and RT4 cell lysates were clarified by centrifugation. The protein concentration of the supernatants was determined with the BCA protein assay (Thermo Scientific). 10 µg of proteins were resolved by SDS-PAGE in a 4–15% polyacrylamide gels, electrotransferred onto Biorad nitrocellulose membranes and analyzed by incubation with primary antibodies against PPARγ (Abcam #ab41928, used at 1/1000) and β-actin (Sigma Aldrich #A2228, used at 1/25,000). Horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling Technology # 7074, used at 1/3000) was used as the secondary antibody. Protein loading was checked by staining the membrane with Amido Black after electroblotting. Uncropped scans of the western blot are supplied Supplementary Fig. 15 .
3
Quantification of Darpp-32 and t-Darpp Proteins
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Mouse tissue and cultured cell lysates were collected on ice in RIPA buffer from Thermo Scientific (Waltham, MA) supplemented with 1× protease inhibitor cocktail from Roche Applied Science (Indianapolis, IN). Protein concentration was determined by RC DC protein assay purchased from Bio-Rad Laboratories (Hercules, CA). 30 μg of protein from each sample was loaded onto a 12% SDS-PAGE gel for protein separation and proteins were transferred to a nitrocellulose membrane. 5% non-fat dry milk was used for a blocking buffer and for primary antibody incubation. Primary antibodies included: an antibody that recognizes both Darpp-32 and t-Darpp (#H62) from Santa Cruz Biotechnology (Santa Cruz, CA) and antibodies to α-Tubulin (#T5168) and β-Actin (#A4700) from Sigma-Aldrich Corporation (St. Louis, MO). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies from Cell Signaling Technology (Danvers, Massachusetts). Secondary antibody was detected using an ECL Plus kit from Thermo Fisher Scientific. Protein expression was quantified using ImageJ software and expressed as relative density, normalized to loading control values. Mammary tissue was arbitrarily considered positive for protein expression when the relative density was greater than 0.5.
4
Antibody Panel for Protein Detection
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The following antibodies were used: anti-FLAG (F1804; Merck), anti-keratin 5 (RM-2106-S0; Thermo Fisher Scientific), anti-keratin 8 (ab53280; Abcam, Cambridge, UK), anti-keratin 14 (MS-115-P0; Thermo Fisher Scientific), anti-keratin 18 (MS-142-P0; Thermo Fisher Scientific), anti-SON (HPA023535; Merck), anti-FAM83H (HPA024604; Merck), anti-CK1α (sc-6477; Santa Cruz Biotechnology, CA, USA), anti-GAPDH (2275-PC-100; R&D Systems, MN, USA), and anti-SC-35 (S4045; Merck). Alexa Fluor 488-conjugated donkey anti-mouse IgG (A-21202) and donkey anti-rabbit IgG (A-21206), Alexa Fluor 568-conjugated donkey anti-mouse IgG (A10037) and donkey anti-rabbit IgG (A10042), Alexa Fluor 594-conjugated donkey anti-goat IgG (A-11058), and Alexa Fluor 647-conjugated donkey anti-mouse IgG (A-31571) were purchased from Thermo Fisher Scientific. Horseradish peroxidase-conjugated anti-mouse IgG (#7076; Cell Signaling Technology, MA, USA) and anti-rabbit IgG (711-035-152; Jackson Immunoresearch, PA, USA) antibodies were used for Western blotting.
5
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed in the lysis buffer (50 mM HEPES (pH 7.4), 200 mM KCl, 1 mM EGTA, 1 mM MgCl2, 10% glycerol, 10% NP-40, 0.5 mM DTT, 0.5 μM microcystin, and 10 μg/mL each of leupeptin, pepstatin, aprotinin, and PMSF). For Western blot analysis, the lysates were separated through SDS-PAGE and transferred to a PVDF membrane (Millipore, Inc). The membrane was incubated with primary antibodies for overnight at 4 °C and secondary antibodies for 1 h at room temperature. Horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling Technology Inc) and anti-rabbit IgG (Enzo Inc.) were used as secondary antibodies for immunoblotting.
6
Western Blot Analysis of HDPC Signaling
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HDPCs (5 × 105) were seeded in 100 mm dishes and incubated at 37°C for 24 h. Then, cells were treated with PG (0-10 μM) in the presence or absence of 20 μM LY294002, 40 μM CHX, 5 μM CHIR99021, 0.1 mM H2O2, or 2 μM MG132 at 37°C. The drug-treated cells were lysed using cell lysis buffer (#4719964001; Roche; Merch KGaA) containing phosphatase inhibitor cocktail (#4906845001; Roche; Merch KGaA). The protein concentration was quantified using Pierce BCA Protein Assay Kit (#23225; Thermo Fisher Scientific). Twenty micrograms of protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes (#88018; Thermo Fisher Scientific). The membranes were incubated with the corresponding primary antibodies overnight at 4°C, and then horseradish peroxidase-conjugated anti-mouse IgG (#7076S; Cell Signaling Technology; CST; USA) or anti-rabbit IgG (#7074S; CST) secondary antibodies were incubated for 2 h at room temperature. The blots were detected using Clarity Western ECL substrates (#1705061; Bio-Rad Laboratories, USA) and the intensities of the protein bands were analyzed with ImageJ software version 1.53t (National Institutes of Health). The following primary antibodies and dilution are listed in Table 2 .
7
Western Blot Analysis of Liver Markers
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The liver tissues and cells were lysed in lysis buffer (Sigma-Aldrich) supplemented with Phosphatase Inhibitor Cocktail II (A. G Scientific Inc., San Diego, CA, USA) and Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total proteins were loaded onto SDS-PAGE gels and transferred to PVDF membranes (BIO-RAD, Hercules, CA, USA), and then incubated overnight at 4 °C with one of the following primary antibodies as indicated: rabbit anti-Col Ⅰ) (1:1000; Novus, Centennial, CO, USA), mouse anti-α-SMA (1:1000; Dako), mouse anti-VEGF (1:500; Novus), rabbit anti-ALB (1:1000; Novus), mouse anti-Cyclin D1 (1:1000; Abfrontier, Seoul, Korea), rabbit anti-hepatic nuclear factor α (HNF1α; 1:1000; Abcam Inc., Cambridge, MA, USA), mouse anti-IL-6 (1:1000; Abcam), rabbit anti-glycoprotein 130 (gp130; 1:250; Santa Cruz Biotechnology), rabbit anti-phosphorylated Stat3 (1:500; Cell Signaling), mouse anti-Stat3 (1:500; Cell Signaling), and rabbit anti-GAPDH (1:3000; Abfrontier). The membranes were washed and then reacted with a secondary antibody (horseradish peroxidase-conjugated anti-mouse IgG (1:5000; Cell Signaling) or anti-rabbit IgG (1:10,000; Cell Signaling) for 1 h at RT. The membranes were incubated using enhanced chemiluminescence reagents (Thermo Fisher Scientific., Waltham, MA, USA).
8
Immunoblot Analysis of HsfB2b-eGFP
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For immunoblot assay, the seedlings of pHsfB2b::HsfB2b-eGFP were harvested at each time point. Total proteins were prepared from 100 mg of harvested samples in protein extraction buffer (50 mM Tris–Cl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM 1,4-Dithiothreitol (DTT), 1Χ complete Mini, and EDTA-free protease inhibitor cocktail (Roche). Total proteins were separated by sodium-dodecyl sulfate (SDS)-PAGE. For phosphatase assay, total proteins were treated with or without alkaline phosphatase (Thermo scientific, EF0652) for 1 h at 37 °C, then separated by SDS-PAGE. The proteins were transferred to PVDF membranes (Amersham Biosciences) and probed with anti-GFP (Clontech, JL-8, 1:10,000 dilution) or anti-myc (Santa Cruz Biotechnology, sc-40, 1:10,000 dilution) antibodies overnight at 4 ℃. The samples were then probed with horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling, #7076, 1:10,000 dilution) antibodies at room temperature. The signals were detected using ImageQuant LAS 4000 (GE Healthcare) with WesternBrightTM Sirius ECL solution (Advansta).
9
HMGB1 Signaling Pathway Regulation
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Rapamycin, LY294002, FPS-ZM1 and U0126 were purchased from Selleck Chemicals (Houston, TX, USA) and recombinant human HMGB1 (rhHMGB1) was provided by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Human HMGB1 ELISA kits were purchased from CUSABIO (Wuhan, China), EdU proliferation detection kits were purchased from RiboBio Co., Ltd. (Guangzhou, China), primary antibodies against β-actin, p-Akt (Ser473), Akt, p-mTOR (Ser2448), mTOR, p-P70S6K (Thr421/Ser424), P70S6K, p-S6 (Ser240/244), S6, p-ERK (Thr202/Tyr204), ERK, p-P90RSK (Ser380), P90RSK-1, p-CREB (Ser133s), CREB, cyclin D1, cyclin E1, TLR2 and RAGE were purchased from Cell Signalling Technology (Beverly, MA, USA). HMGB1 antibody was purchased from AbCam (Cambridge, UK) and TLR4 antibody was provided by ABclonal Biotechnology Co., Ltd (Wuhan, China). E-cadherin, N-cadherin, MMP-9 and MMP-2 antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies coupled to IRDye800 fluorophore for use with the Odyssey Infrared Imaging System were purchased from LI-COR Biosciences (Lincoln, Nebraska, USA). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).
10
Src Family Kinases Signaling Dynamics
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The following antibodies were used: anti-Src (05–184; Merck, Darmstadt, Germany), anti-Yes (610375; BD Biosciences, San Jose, CA, USA), anti-Lyn (sc-7274; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Csk (610080; BD Biosciences), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2275-PC-100; R&D Systems, Minneapolis, MN, USA), anti-p-Tyr (05–1050; Merck), anti-Src family phospho-Y418 (p-SFKs A-loop; ab40660; Abcam, Cambridge, UK), anti-p-Src Y530 (sc-166860; Santa Cruz Biotechnology), anti-p-Lyn Y508 (CSB-PA000691; Cusabio, Wuhan, China)), anti-Fyn (sc-434; Santa Cruz Biotechnology), anti-E-cadherin (#3195; Cell Signaling Technology, Danvers, MA, USA), anti-vimentin (V6630; Sigma-Aldrich), anti-Cbl-b (sc-8006; Santa Cruz Biotechnology) and anti-c-Cbl (sc-1651; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-mouse IgG (#7076; Cell Signaling Technology) and anti-rabbit IgG (711-035-152; Jackson Immunoresearch, West Grove, PA, USA) antibodies were used for Western blotting.
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