Western blot studies were performed as previously described6 (link). After the LPS and LECT2 stimulation, treated cells were collected and lysed in 1 × RIPA lysis buffer (Upstate Biotechnology) with a Complete Mini EDTA-free cocktail tablet (Roche Diagnostics) and PhosSTOP phosphatase inhibitor cocktail tablets (Roche Diagnostics). Protein samples were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes, using an iBlot gel transfer system (Invitrogen). Membranes were blocked and then they were probed with antibodies for 16 h (Phospho-SAPK/JNK (Thr183/Tyr185) Antibody (Cell Signaling, Danvers, MA), SAPK/JNK Antibody (Cell Signaling, Danvers, MA), Phospho-p38 MAPK (Thr180/Tyr182) Antibody (Cell Signaling, Danvers, MA), p38 MAPK Antibody (Cell Signaling, Danvers, MA), Phospho-p44/42 MAPK(Erk1/2) (Thr202/Tyr204) Antibody (Cell Signaling, Danvers, MA), p44/42 MAPK(Erk1/2) Antibody (Cell Signaling, Danvers, MA) Phospho-SEK1/MKK4 (Ser257) Antibody (Cell Signaling, Danvers, MA), SEK1/MKK4 Antibody (Cell Signaling, Danvers, MA) Afterward, membranes were washed and then incubated with anti-rabbit IgG horseradish peroxidase-linked antibody (Cell Signaling) for 1 h. Protein bands were visualized with ECL Prime Western blotting detection reagent (GE Healthcare UK Ltd.).
Phospho p38 mapk thr180 tyr182 antibody
Manufactured by Cell Signaling Technology
The Phospho-p38 MAPK (Thr180/Tyr182) Antibody is a specific antibody that recognizes the phosphorylated form of p38 MAPK at the Thr180 and Tyr182 residues. The antibody is designed for the detection of the activated form of p38 MAPK in various applications, such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
P38 mapk antibody, by Cell Signaling Technology (2 mentions)
Iblot gel transfer system, by Thermo Fisher Scientific (1 mentions)
P44 42 mapk erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204 antibody, by Cell Signaling Technology (1 mentions)
Complete mini edta free cocktail tablet, by Roche (1 mentions)
Phospho sapk jnk thr183 tyr185 antibody, by Cell Signaling Technology (1 mentions)
Sapk jnk antibody, by Cell Signaling Technology (1 mentions)
Anti rabbit igg horseradish peroxidase linked antibody, by Cell Signaling Technology (1 mentions)
Phosstop phosphatase inhibitor cocktail tablet, by Roche (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Yn 20, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ipvh00010, by Cytiva (1 mentions)
Novex tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Gapdh 14c10 rabbit mab, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phospho akt ser473 antibody, by Cell Signaling Technology (1 mentions)
Mtor antibody, by Cell Signaling Technology (1 mentions)
Phospho erk antibody, by Cell Signaling Technology (1 mentions)
Electrochemiluminescence kit, by Cytiva (1 mentions)
5 protocols using phospho p38 mapk thr180 tyr182 antibody
1
Western Blot Analysis of MAPK Signaling
2
Protein Extraction and Western Blot Analysis
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Protein extraction and western blot analysis was carried out as previously described [6 (link)] using a phospho-p38 MAPK (Thr180/Tyr182) antibody to detect the SakA phosphorylated form, a phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody to detect the MpkA phosphorylated form (Cell Signaling Technology), and a γ-tubulin antibody as a reference (yN-20) (Santa Cruz biotechnology, Inc.). Every experiment was repeated at least three times, using independent biological replicates. Additional information is reported in S1 Text .
3
Western Blot Analysis of Signaling Pathways
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One million U937 cells were stimulated, washed in PBS, and lysed in buffer (4% SDS, 40 mM HEPES [pH 7.4, 10 mM DTT] supplemented with protease inhibitors [Sigma‐Aldrich, 4693159001]). Samples were centrifuged (16,000 g, 10 min), Li‐LDS sample buffer was added to a final concentration of 1×, and the supernatant was incubated (5 min, 95°C). Proteins were separated on 12% Novex Tris‐glycine gels (Thermo Fisher Scientific, XP00120BOX) and transferred onto PVDF membranes (Merck Millipore, IPVH00010) or Nitrocellulose membranes (Amersham, 10600002). Membranes were blocked in 5% BSA in PBST, and antibodies were diluted in 2% BSA in PBST. Antibodies used for immunoblotting were as follows (diluted 1:1,000): phospho‐p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling, 9211), GAPDH (14C10) rabbit mAb (Cell Signalling, 2118), p38 MAPK (R&D, AF8691), ARHGEF18 (Sigma, HPA042689), MAP3K7 (R&D, MAB5307), FOSB (R&D, AF2214) and anti‐rabbit IgG, HRP‐linked antibody (Cell Signaling, 7074).
4
Western Blot Analysis of EIF3C Expression
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In order to estimate EIF3C expression status in MDA-MB-231 and BT474 cells western blotting analysis was performed. The proteins were probed by anti-EIF3C Antibody (1: 1,000; #HPA050112, Sigma), AKT Antibody (1: 1,000; #9272, Cell Signaling Technology), Phospho-Akt (Ser473) Antibody (1: 1,000; #9271, Cell Signaling Technology), ERK Antibody(1: 1000; #9102, Cell Signaling Technology), phospho-ERK Antibody (1: 1,000; #9101, Cell Signaling Technology), p38 MAP Kinase Antibody (1: 1,000; #9212, Cell Signaling Technology), phospho-p38 MAPK(Thr180/Tyr182) Antibody (1: 1,000;#9216S, Cell Signaling Technology),mTOR Antibody (1: 1,000; #2971, Cell Signaling Technology), Raptor Antibody (1: 1,000; #2280, Cell Signaling Technology) and detected by Electrochemiluminescence (ECL) kit (Amersham). GAPDH and β-actin were used as normalized controls (Santa Cruz Biotechnology, USA).
5
Protein Expression Analysis by Western Blot
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Cells or tissues were lysed using RIPA buffer (Cell Signaling Technology, 9806). Protease Inhibitor Cocktail (Cell Signaling Technology, 5871) was added to the lysates. Proteins were separated by SDS–PAGE and transferred to nitrocellulose membranes to assess abundance by primary antibody detection. The primary antibodies used were Phospho‐CaMKII (Thr286) Rabbit mAb (1:1,000; Cell Signaling Technology, cat# 12716), CaMKII (pan) Rabbit mAb (1:1,000; Cell Signaling Technology, cat# 4436), Phospho‐Elk‐1 (Ser383) Antibody (1:1,000; Cell Signaling Technology, 9181), Elk‐1 Antibody (1:1000; Cell Signaling Technology, cat# 9182), p38 MAPK Antibody (1:1,000; Cell Signaling Technology, cat# 9212), Phospho‐p38 MAPK (Thr180/Tyr182) Antibody (1:1,000; Cell Signaling Technology, cat# 9211), Anti‐c‐Fos (phosphor T232) antibody (1 μg/ml; Abcam, cat# ab43175) and Anti‐c‐Fos (phosphor T232) antibody (1 μg/ml; Abcam, cat# ab90289), Anti‐GRO alpha antibody (4 μg/ml Abcam, cat# ab86436), and Monoclonal Anti‐β‐Actin (1 μg/ml; Sigma, cat# A1978). Signals were visualized using ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106). Band intensities were quantified using the ImageJ software for grayscale statistics.
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