TMTH was supplied by Gyeongnam Agricultural Research and Extension Services, Jinju, South Korea. DNCB was procured from Sigma-Aldrich (St. Louis, MO, USA) and olive oil was obtained from a supermarket (South Korea). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062), TRAF6 (sc-8409), TNF-α (sc-52746), cyclooxygenase-2 (COX2; sc-376861) and inducible nitric oxide synthase (iNOS; sc-7271) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against MyD88 (4283S), IRAK4 (4363T), p-ERK (4377S), ERK (4695S), p-JNK (4668S), JNK (9258S), p-p38 (9215S) and p-38 (9212S) were obtained from Cell Signaling Technology (Danvers, MA, USA).
Sc 8409
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-8409 is a laboratory instrument designed for sample preparation and analysis. It is a centrifuge capable of separating components of a mixture based on their density and size. The core function of Sc-8409 is to facilitate the isolation and purification of various biological samples, such as cells, organelles, or macromolecules, for further study and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Xpert protease inhibitor cocktail solution 100, by GenDEPOT (2 mentions)
Sc 52746, by Santa Cruz Biotechnology (1 mentions)
Cox2 sc 376861, by Santa Cruz Biotechnology (1 mentions)
Inos sc 7271, by Santa Cruz Biotechnology (1 mentions)
Gapdh sc 365062, by Santa Cruz Biotechnology (1 mentions)
Ab181553, by Abcam (1 mentions)
Gtx109242, by GeneTex (1 mentions)
Gtx130246, by GeneTex (1 mentions)
Yt4689, by Immunoway (1 mentions)
Df6251, by Affinity Biosciences (1 mentions)
Peroxidase, by GE Healthcare (1 mentions)
Amersham hybond p 0, by GE Healthcare (1 mentions)
Ecl prime detection kit, by GE Healthcare (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
β actin clone ac 15, by Merck Group (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Ab238, by Abcam (1 mentions)
Ab109429, by Abcam (1 mentions)
Ab76424, by Abcam (1 mentions)
C fos, by Cell Signaling Technology (1 mentions)
7 protocols using sc 8409
1
Topical Skin Inflammation Modulation Protocol
2
Protein Expression in Ischemic Penumbra
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Proteins from ischemic penumbral tissues and HAPI cells were used for Western blot analysis. SDS-PAGE was performed, and the resolved proteins were transferred onto polyvinylidene fluoride (PVDF) membranes by electroblotting. The membranes were blocked at Tris–buffered saline (TBST) containing 5% non-fat milk powder for 2 h and then incubated with the following primary antibodies overnight at 4°C: Arg1 (1:500, GTX109242, GeneTex), CD163 (1:500, WH112776, ABclonal), iNOS (1:500, GTX130246, GeneTex), CD86 (1:500, WH141312, ABclonal), P62 (1:200, WH146703, ABclonal), IRF5 (1:1000, ab181553, Abcam), TRAF6 (1:200, sc-8409, Santa Cruz), IKKαβ (1:200, BM4499, Boster), IL-10 (1:200, 20850-1-AP, Proteintech), IL-4 (1:500, 66142-1-lg, Proteintech), TNF-α (1:200, YT4689, ImmunoWay), and IL-1β (1:500, DF6251, Affinity). After being washed with TBST 3 times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse secondary antibodies for 2 h at RT. β-actin was used as a loading control. Immunoreactive bands were detected by an enhanced chemiluminescence (ECL) detection system. The gray values of the protein bands were quantified by Image Lab.
3
Western Blot Analysis of Aortic Proteins
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Protein lysates were obtained from thoracic aortas of BM miR-146a-/-and BM wt transplanted mice fed with a HFD for 20 weeks. Tissue homogenization was performed using TissueLyser (Qiagen). Normalization was based on the amount of protein loaded. Total protein (20 μg) was separated by SDS-PAGE (8%) in reducing conditions. Gels were transferred onto PVDF membranes (Amersham Hybond P 0.45, GE Healthcare, Barcelona Spain) and blocked for 1 hour in 5% w/v BSA-TBS. Membranes were incubated overnight at 4°C with primary antibodies against human Traf6 (#sc-8409, 1:1,000, Santa Cruz, Heidelberg, Germany), Irak1 (D51G7, 1:1,000, Cell Signalling Technology, Leiden, The Netherlands), and β-actin (AC-15 clone, 1:5,000, Sigma-Aldrich, Madrid, Spain). After washing, membranes were incubated with secondary antibodies labeled with peroxidase (1:10,000, GE Healthcare, Barcelona, Spain). ECL Prime Detection Kit and ImageQuant LAS 4000 Imager (GE Healthcare, Barcelona, Spain) were used for protein detection. Densitometric analyses were performed with ImageJ software (http://rsb.info.nih.gov/ij/ ).
4
Western Blotting and Immunoprecipitation Assay
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For western blotting, total protein was extracted from the cultured cells using RIPA lysis and extraction buffer (Thermo Fisher Scientific) and separated on a 12% SDS–PAGE polyacrylamide gel.
The primary antibodies used in this study were IFITM3 (1:1,000 dilution, ab109429, Abcam), TRAF6 (1:100, sc-8409, Santa Cruz Biotechnology), IRAK1 (1:200, ab238, Abcam), p-ERK (1:800, #4376, Cell Signaling Technology), c-Fos (1:20,#4384, Cell Signaling Technology), c-Myc (1:100, 395R16, Cell Marque), and survivin (1:50, ab76424, Abcam). β-actin served as the internal control.
For the immunoprecipitation (IP) assay, the protein extracts from cultured cells were prepared using IP lysis buffer (50 mM Tris–Cl [pH 7.4], 0.5% NP-40, 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, 2 mM EGTA) supplemented with protease/phosphatase inhibitor mixtures (Roche), then centrifuged (12,000 g for 10 minutes at 4℃). The IP assay was performed as previously described [18 (link)].
The primary antibodies used in this study were IFITM3 (1:1,000 dilution, ab109429, Abcam), TRAF6 (1:100, sc-8409, Santa Cruz Biotechnology), IRAK1 (1:200, ab238, Abcam), p-ERK (1:800, #4376, Cell Signaling Technology), c-Fos (1:20,#4384, Cell Signaling Technology), c-Myc (1:100, 395R16, Cell Marque), and survivin (1:50, ab76424, Abcam). β-actin served as the internal control.
For the immunoprecipitation (IP) assay, the protein extracts from cultured cells were prepared using IP lysis buffer (50 mM Tris–Cl [pH 7.4], 0.5% NP-40, 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, 2 mM EGTA) supplemented with protease/phosphatase inhibitor mixtures (Roche), then centrifuged (12,000 g for 10 minutes at 4℃). The IP assay was performed as previously described [18 (link)].
5
Analyzing EZH2 Stability in Cell Lysates
6
Western Blot Analysis of Hepatitis C Viral Proteins
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Cells were lysed in lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 5 mM EDTA, 0.1% NP40, and Xpert protease inhibitor cocktail Solution 100× (catalog no. p3100-010; GenDEPOT, Barker, TX, USA). Protein concentration was determined by Bradford assay. A 10–12% SDS-polyacrylamide gel was run under standard electrophoresis conditions, with 30 µg of total protein loaded in each lane. Separated proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA) at 100 V for 100 min, and incubated with primary antibodies in blocking solution (3% BSA). Proteins were visualized using the Western blotting luminol reagent (catalog no. sc-2048; Santa Cruz, Dallas, TX, USA). The following antibodies were used: mouse anti-NS5B; 5B-3B1 (Enzo Life Science; Switzerland), mouse anti-NS3; sc-69938, mouse anti-NS4B; sc-52416, mouse anti-NS5A; sc-65458, goat anti-Lamin B; sc-6216, mouse anti-α-Actinin; sc-17829, mouse anti-β-actin; sc-47778, mouse anti-TRAF6; sc-8409 (Santa-Cruz, Dallas, TX, USA), rabbit anti-NF-kB(P65); 8242s, rabbit anti-p62; 8025s, and rabbit anti-LC3B; 2775s (Cell signalling, Danvers, MA, USA). All first antibody dilution was 1:1,000. Secondary antibodies used goat anti-mouse IgG; M32607, goat anti-rabbit IgG; 31460 (Invitrogen, Waltham, MA, USA), rabbit anti-goat IgG; SA007-500 (GenDEPOT, Barker, TX, USA). All secondary antibody dilution was 1:10,000.
7
TRAF6 Immunoprecipitation and Western Blotting
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Treated or non-treated cells were lysed in lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 12 mM sodium deoxycholate, 1% Triton x-100, 0.1% SDS and Xpert protease inhibitor cocktail Solution 100× (catalog no. p3100-010; GenDEPOT, Barker, TX, USA), and cell lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4 °C. The lysates (1 mg) were incubated with anti-TRAF6 (4 ug) for 24 h at 4 °C. Protein-A/G-conjugated agarose beads were added, followed by incubation for 4 h at 4 °C. Beads were washed three times with 1X TBST. Immunopellets were boiled with SDS-PAGE sample buffer and resolved by electrophoresis. The following antibodies were used: mouse anti-TRAF6; sc-8409, mouse anti-β-actin; sc-47778 (Santa-Cruz, Dallas, TX, USA), and rabbit anti-p62; 8025s (Cell signalling, Danvers, MA, USA).
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