Protein extraction was carried out by lysing cells in PBS buffer containing 1% Triton X-100 with added protease and phosphatase inhibitors (Roche). Protein content was determined using Bradford assay (Pierce). Approximately 25 to 70 μg of total protein was resolved on 8, 10 or 12% SDS-PAGE gel before transferring onto PVDF membrane (Millipore) for detection using ECL reagent (Pierce). The following antibodies were used: anti-phospho-JNK, anti-JNK, anti-phospho-MKK4, anti-MKK4, anti-FTH1 (Cell Signaling Technology), anti-TRX (Santa Cruz), anti-LC3B (Abgent), anti-SOD2 (Abcam), anti-actin and HRP-conjugated anti-rabbit and anti-mouse (Sigma).
Anti mkk4
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-MKK4 is a laboratory reagent that targets the mitogen-activated protein kinase kinase 4 (MKK4) protein. MKK4 is an important enzyme involved in cellular signaling pathways. This product can be used to detect and study the expression and activity of MKK4 in various experimental systems.
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Lab products found in correlation
Anti phospho mkk4, by Cell Signaling Technology (3 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti fth1, by Cell Signaling Technology (1 mentions)
Anti trx, by Santa Cruz Biotechnology (1 mentions)
Anti lc3b, by Abcepta (1 mentions)
Anti sod2, by Abcam (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti p p38, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Anti phospho ikkα β ser176 180, by Cell Signaling Technology (1 mentions)
Anti tak1, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Merck Group (1 mentions)
Anti phospho p38 t180 y182, by Cell Signaling Technology (1 mentions)
Anti iκb, by Cell Signaling Technology (1 mentions)
Anti tak1, by Santa Cruz Biotechnology (1 mentions)
Anti ask1, by Cell Signaling Technology (1 mentions)
5 protocols using anti mkk4
1
Protein Extraction and Western Blot
2
Western Blot Analysis of MAPK Signaling
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After HWY336 treatments, total protein (50 µg) of cell lysate was separated by 10% SDS-PAGE and transferred to a PVDF membrane. Specific proteins were detected using the following primary antibodies: anti-JNK, anti-p-JNKs, anti-p38, anti-p-p38, anti-MKK4, and anti-p-MKK4 (Cell Signaling Technology, Inc.).
3
Signaling Pathway Characterization Protocol
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Anti–TAK‐1 (catalog no. D94D7), anti–MKK‐4 (catalog no. 9152S), anti–phospho–MKK‐4T261 (catalog no. 9151S), anti‐IκB (catalog no. 4814S), anti–phospho‐IκB, Ser32/36 (catalog no. 9246), anti–phospho–p38‐T180/Y182 (catalog no. 9211S), anti–phospho–TAK‐1‐T184/187 (catalog no. 45085), anti–phospho–IKKα/β‐Ser176/180 (catalog no. 16A6), and anti–phospho–activating transcription factor 2 (ATF‐2)–Thr71 (catalog no. 9221) were from Cell Signaling Technology. Anti–phospho–JNK‐pTpY183/185 (catalog no. 44682G) was obtained from Invitrogen. Anti–T‐ERK (catalog no. sc‐94), anti–NF‐κB–p65 (catalog no. Sc‐372), Anti–TAK‐1 (catalog no. sc‐7162), and anti–phospho–TAK‐1‐S192 (catalog no. sc‐130219) were from Santa Cruz Biotechnology. Anti‐ubiquitin–Lys‐63–specific antibody (catalog no. 05‐1308) was from Millipore. Myelin basic protein (MBP; catalog no. M1891) and anti–phospho‐ERK (catalog no. M9692) were from Sigma. A ubiquitin chain restriction enzyme analysis (UbiCREST) kit (catalog no. K‐400) was obtained from R&D Systems, and γ32P‐ATP was obtained from PerkinElmer. TAK‐1 inhibitor 5z‐7‐oxozeanol (catalog no. 3604) was from Tocris.
4
Protein Extraction and Western Blot Analysis
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Preparation of total protein extracts from mice liver or cells was performed following the procedure described previously [36 (link)]. The extracted proteins and immunoprecipitates were separated by SDS-PAGE 10% or 12% polyacrylamide gel and then electrically transferred to a PVDF membrane. After blocking with 5% (w/v) BSA in TBST at room temperature for 1 h, the membranes were then incubated with an appropriate specific primary antibody (Anti-JNK1/2, anti-phospho JNK1/2, anti- MKK4, anti-phospho MKK4, anti-ASK1, anti-phospho ASK1, anti-Ubiquitinin, Cell Signaling Technology, Boston; Anti-mono and dimethyl Arginine, Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:15,000; Sunshine Biotechnology) and detected by enhanced chemical luminescence kit (Thermo scientific, Hudson, NH, USA). The quantification of the bands was performed by the Gel Analysis V2.02 Software (Clin Science Instruments, Shanghai, China).
5
Antibody Procurement for Cell Signaling
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