We stimulated macrophages (2 × 106/mL) from the WT and CD14 KO mice with ArtinM (2.5 µg/mL), LPS (1 µg/mL), P3C4 (1 µg/mL), LTA (1 µg/mL), or medium for 15 and 45 min. Subsequently, the cells were washed in cold PBS and lysed with RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0) supplemented with protease and phosphatase inhibitors. Lysate samples were analyzed by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes following a protocol described elsewhere76 (link). The membrane was probed with anti-IκB-α (1:1000; clone C-21, Santa Cruz, CA, USA), anti-p- IκB-α (1:1000; clone B-9, Santa Cruz, CA, USA) and anti-β-actin (1:1000; clone C4, Santa Cruz, CA, USA). We then carried out a second staining with peroxidase-conjugated secondary antibody (1:3000). The membrane was incubated with ECL reagent for 1 min to detection of protein using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, USA).
Clone c4
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Clone C4 is a laboratory instrument designed for the isolation and purification of specific DNA or RNA sequences. It utilizes a proprietary cloning technology to selectively amplify and extract target genetic material from complex samples. The core function of the Clone C4 is to provide researchers with a reliable and efficient tool for molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti total enos, by BD (2 mentions)
Anti p s1177 enos, by BD (2 mentions)
Horse radish peroxidase conjugated donkey anti mouse or anti rabbit secondary antibody, by Jackson ImmunoResearch (2 mentions)
Anti total enos, by Santa Cruz Biotechnology (2 mentions)
Clone 3 enos nos type 3, by Santa Cruz Biotechnology (2 mentions)
Anti piezo1, by Proteintech (2 mentions)
Phosstop phosphatase inhibitor, by Roche (2 mentions)
Minicomplete protease inhibitors, by Roche (2 mentions)
Anti iκbα, by Santa Cruz Biotechnology (1 mentions)
Anti p iκb α, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Clone b 9, by Santa Cruz Biotechnology (1 mentions)
Benchmark xt, by Roche (1 mentions)
Vector novared peroxidase hrp substrate kit, by Vector Laboratories (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab9116, by Abcam (1 mentions)
Sc 789, by Santa Cruz Biotechnology (1 mentions)
Sc 134036, by Santa Cruz Biotechnology (1 mentions)
Westernbright sirius chemiluminescent hrp substrate, by Advansta (1 mentions)
10 protocols using clone c4
1
Macrophage Signaling Pathway Analysis
2
Evaluating BAP1 Protein Expression
3
Automated and Manual BAP1 Immunohistochemistry
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Immunohistochemistry was performed on 4-μm-thick FFPE sections using an automated immunostainer (Benchmark XT, Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions. Sections were immunostained with a primary monoclonal antibody against BAP1 (clone C4, 1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) using 3,3-diaminobenzidine (DAB) as chromogen. Selected cases that showed a staining result that was difficult to interpret including strongly pigmented tumors (patients #1–8, #12–16, #22–24) were manually stained (clone C4, 1:50 dilution, Immunologic, Duiven, the Netherlands) according to the manufacturer’s instructions. The VECTOR NovaRED Peroxidase (HRP) Substrate Kit was used for visualization (Vector Laboratories, USA, Catalogue Number SK-4800). Nuclei of endothelial and lymphocytic cells in the slides served as positive internal control for BAP1 protein expression. The staining results were scored independently by three pathologists (HK, DC, WB). The percentage of positive tumor cell nuclei was scored only in areas with positive internal controls.
4
Western Blot Analysis of Protein Expression
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Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Equal amount of protein lysate was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore) using fast Turbo Transfer (Bio-rad). After blocking in 5% skimmed milk for 1 h, the appropriate primary antibody was added: anti-ADAR2 (RED1) (1:1000, ab64830, Abcam), anti-SRp30c(SRSF9) (1:1000, sc-134036, Santa Cruz), anti-FLAG (1:2000, Clone M2, Sigma), anti-cMyc (1:1000, sc-789, Santa Cruz), anti-V5 (1:2000, ab9116, Abcam) or anti-β-actin (1:1000, clone C4, sc-47778, Santa Cruz). Primary antibodies were incubated overnight in the cold room. After washing with phosphate-buffered saline (PBS)/0.2% Tween-20 (PBST), a secondary antibody, namely horse-radish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000, NA934V, Amersham) or HRP-conjugated anti-mouse IgG (1:5000, NA931V, Amersham), was added for 1 h. After washing with PBST, signals were detected using the WesternBright Sirius Chemiluminescent HRP Substrate (Advansta).
5
Western Blot Analysis of Protein Targets
6
Immunohistochemical Detection of BAP1 in Tumors
7
Western Blot Analysis of Protein Targets
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Cells were transfected and harvested in lysis buffer containing 10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5 % NP-40, MiniComplete protease inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche). Equal protein amounts were loaded on 8 % gels and resolved by electrophoresis. Samples were transferred to PVDF membranes and labeled overnight with primary antibody: anti-β-actin (0.2 μg.mL−1, Santa Cruz); anti-total-eNOS (0.2 μg.mL−1, BD Biosciences); anti-p(S1177)-eNOS (0.2 μg.mL−1, BD Biosciences); or anti-Piezo1 (0.3 μg.mL−1, Proteintech). Horse radish peroxidase-conjugated donkey anti-mouse or anti-rabbit secondary antibody (Jackson ImmunoResearch) and SuperSignal Femto detection reagent (Perbio Science) were used for visualization. Densities of protein bands were quantified by ImageJ software. Catalogue information for antibodies: anti-β-actin (0.2 μg.mL−1, Clone C4, sc-47778; Santa Cruz); anti-total-eNOS (0.2 μg.mL−1, Clone 3/eNOS/NOS Type III, Cat. #610297; BD Biosciences); anti-p(S1177)-eNOS (0.2 μg.mL−1, Cat. #612392; BD Biosciences); or anti-Piezo1 (0.3 μg.mL−1, Proteintech).
8
Western Blot Analysis of Inflammasome Proteins
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For Western blotting 5 × 105 GEN2.2 cells were lysed in Laemmli buffer, heated at 100 °C for 10 min, separated on 10 or 15% SDS-PAGE then electro-transferred to nitrocellulose membranes (Bio-Rad Laboratories GmbH, Munich, Germany). Non-specific binding sites were blocked with 5% non-fat dry milk diluted in TBS Tween buffer (50 mM Tris, 0.5 M NaCl, 0.05% Tween-20, pH 7.4). Membranes were probed with the anti-NLRP3 (clone D2P5E, Cat. No. 13158S), anti-ASC (clone E1E3I; Cat. No. 13833S), anti-IL-1β (clone D3U3E; Cat. No. 12703S) and anti-caspase-1 (clone D7F10; Cat. No. 3866S) (all from Cell Signaling). Beta-actin was used as a loading control (Santa Cruz Biotechnology, clone C4; Cat. No. sc-47778). The bound primary antibodies were conjugated with anti-mouse (Bio-Rad, Cat. No. 1721011) or anti-rabbit (GE Healthcare, Cat. No. NA934) horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:5000 and 1:10,000, respectively, and were visualized by the ECL system using SuperSignal West Pico or Femto chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, United States) and X-ray film exposure. Densitometric analysis of immunoreactive bands was performed using Image Studio Lite Software version 5.2 (LI-COR Biosciences, Lincoln, NE, United States).
9
Comprehensive IHC Protocol for BAP1, p16, MTAP
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Standardized protocol using the DAKO platform was used for IHC on FFPE tissue for BAP1 (1:50 primary, Clone C4; Santa Cruz Biotech, TX), p16 (1:400 primary, Clone JC2; Cell Marque, CA), and MTAP (1:100 primary, Clone 2G4; Abnova, Taipei, Taiwan). IHC for H3 K27me3 (1:600 primary, Clone C36B11; Cell Signaling, MA) was performed on the Leica BOND platform.
10
Immunofluorescence Assay for Transfected Cells
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H1299 cells were transfected using the Qiagen Effectene kit according to the manufacturer’s manual in a 12-well plate seeded on glass slides. Twenty-four hour after transfection, cells were washed three times with PBS, fixed with 3.7% formaldehyde for 10 min and washed again three times with PBS. Cells were permeabilized by permeabilization buffer (PBS with 0.1% Triton X-100) for 15 min and blocked for 30 min (1% BSA, 0.1% Tween-20 in PBS). Anti-Myc (1:500, A190-104A, Bethyl) and anti-β-actin (1:100, clone C4, Santa Cruz) antibody were incubated overnight at 4 °C in blocking buffer. Slides were washed three times with PBSt (containing 0.1% Tween-20), incubated with secondary antibody (1:1000 in blocking buffer, donkey anti-mouse Alexa 488, donkey anti-goat Alexa 568, Thermo Fisher Scientific) for 1 h at RT in the dark and washed three times. Coverslips were mounted using Mowiol (Carl Roth GmbH) mounting medium, including DAPI (Thermo Fisher Scientific) and dried for 1–2 days. The mounting medium was prepared as described in CSH protocols (http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10255 ). Pictures were taken using the confocal microscope Leica TCS SP5.
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