Tongue cancer cells were pretreated with or without an ERK inhibitor (U0126, Cell Signaling Technology, Danvers, MA, USA), a JNK1/2 inhibitor (JNK-IN-8, Calbiochem, San Diego, CA, USA), or a p38 inhibitor (SB203580, Calbiochem, San Diego, CA, USA) for 2 h and maintained in the presence or absence of DSK for 24 h. Total protein lysates (20 μg) were harvested and subjected to SDS-PAGE analyses [43 (link),44 (link)]. Specific antibodies targeting the following molecules were used for detection: Anti-cleaved Caspase-8 (#9496), Anti-cleaved Caspase-9 (#9505), Anti-cleaved Caspase-3 (#9664), Anti-Caspase-8 (#9746), Anti-Caspase-9 (#9502), Anti-PARP (#9542), Anti-Phospho-Erk1/2 (#4370), Anti-Erk1/2 (#9102), Anti-Phospho-JNK (#4668), Anti-JNK2 (#9258), Anti-c-IAP1 (#7065), and Anti-XIAP (#2045) antibodies from Cell Signaling Technology (Danvers, MA, USA); Anti-Caspase-3 (610323), Anti-phospho-p38 (612281), and Anti-p38 (612168) antibodies from BD biosciences (San Jose, CA, USA); Anti-β-actin (ab8226) and anti-HO-1 (ab68477) antibodies from Abcam (Cambridge, UK); Anti-Nrf2 (GTX55732) antibodies from GeneTex (Irvine, CA, USA); and HRP-conjugated secondary antibodies (Dako Corporation, Carpinteria, CA, USA). Densitometry data of immunoblots were generated and analyzed by ImageJ software.
Anti jnk2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-JNK2 is a primary antibody that specifically recognizes the JNK2 protein. JNK2 is a member of the c-Jun N-terminal kinase (JNK) family, which play a role in cellular stress response pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti erk1 2, by Cell Signaling Technology (3 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (3 mentions)
Anti phospho sapk jnk, by Cell Signaling Technology (2 mentions)
Anti phospho iκbα, by Cell Signaling Technology (2 mentions)
Anti phospho p38 mapk, by Cell Signaling Technology (2 mentions)
Anti p38 mapk, by Cell Signaling Technology (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Anti xiap, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Anti ciap1, by Cell Signaling Technology (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti caspase 8, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Anti gef h1, by Cell Signaling Technology (1 mentions)
Anti rock1, by Cell Signaling Technology (1 mentions)
Irdye800 anti rabbit igg, by LI COR (1 mentions)
7 protocols using anti jnk2
1
Modulation of Tongue Cancer Cell Apoptosis
2
Antibody Validation for ASAP1 and Related Proteins
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A monoclonal mouse anti-ASAP1 antibody was purchased from Abnova. A polyclonal rabbit anti-ASAP1 serum was raised in our lab as previously described (31 (link)). Anti-paxillin and anti-GM130 antibodies were purchased from BD Biosciences, anti-ROCK2 antibody from BD Transduction Laboratories, anti-HABP4 antibody from Bioss USA, anti-Akt, anti-Arf6, anti-GEF-H1, anti-JNK2, anti-PRKAR1A (PKA RI-α/β), anti-ROCK1, anti-SMARCA5 (SNF2H), anti-TIAM1, and anti-VAV2 from Cell Signaling Technology, anti-β-COP antibody from Invitrogen, IRDye680 anti-mouse IgG and IRDye800 anti-rabbit IgG were from LI-COR, and anti-Brag2 (IqSec1) antibody was from Sigma Precision Antibodies.
3
Immunofluorescent Analysis of HIF1α and JNK2
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Paraffin-embedded whole-tissue sections were sectioned at 6 μm thickness and deparaffinized. Antigen retrieval was performed by heating in 0.1 mol/l sodium citrate (pH 6.0) using a microwave. Slides were then incubated overnight at 4 °C with a mixture of two primary antibodies, anti-HIF1α (Sigma) and anti-JNK2 (Cell Signaling), using antibody diluents (IHC World). Slides were rinsed with PBS and then incubated for 1 h with Alexa Fluor 594 or Alexa Fluor 488 (Life Technologies). Slides were mounted with ProLong Gold anti-fade reagent containing 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Cell death was analyzed using the in situ cell death detection kit (Roche), following the manufacturer’s instructions. Confocal images were obtained using a Nikon D-Eclipse C1 confocal system and processed with Nikon EZ-C1 3.90 and Adobe Photoshop software.
4
Lipopolysaccharide-Induced Inflammatory Signaling
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BA (purity ≥98%) was purchased from Psaitong (Beijing, China). Dimethyl sulphoxide (DMSO) and LPS were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies used for western blotting were as follows: anti-iNOS/NOS Type II (#610332; BD Biosciences, San Jose, CA); anti-cyclooxygenase (COX)-2 (#160106), anti-phospho-IKKα/β (S176/180, 16A6, #2697P), anti-phospho-IκBα (Ser32, #2859), anti-IκBα (44D4, #4814), anti-phospho-ERK1/2 (Thr202/Tyr204, #9101), anti-ERK1/2 (#9102), anti-phospho-p38 MAPK (Thr180/Tyr182, #4511), anti-p38 MAPK (#9212), anti-phospho-SAPK/JNK (Thr183/Tyr185, #9251), anti-JNK2 (56G8, #9258) and anti-NF-κB p65 (D14E12, #8242) (Cell Signaling Technology, Beverly, MA); anti-IKKα (CHUK, #A2062; ABclonal Technology, Woburn, MA); anti-β-tubulin (#CW0098A) and anti-GAPDH (#CW0266A) (CWBiotech, Beijing, China); and anti-histone H2B (Santa Cruz Biotechnology, Inc., Dallas, TX).
5
Protein expression analysis by Western blot
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Western blot analysis was performed as described previously [5 (link),6 (link)] using anti-occludin (NBP1-87402) obtained from Novusbio, Total OXPHOS Rodent WB Antibody Cocktail (ab110413) and anti-PGC-1α (ab54481) obtained from Abcam (Cambridge, UK), anti-HSP60 (sc-376240) obtained from Santa Cruz and anti-phospho-SAPK/JNK (Thr183/Tyr185) (#9251), anti-JNK2 (#9258), anti-SOD2 XP (#13141), anti-SIRT3 (#5490), anti-phospho-AKT (Ser473) (#9271), anti-AKT (#9272), anti-IRβ (#3025) and anti-IGF-1Rβ (#3027) antibodies, as well as the secondary antibodies anti-rabbit antibody (#7074) and anti-mouse antibody (#7076) obtained from Cell Signaling (Cambridge, UK). Ponceau staining served as a loading control. Oxyblot analysis was carried out as previously published [33 (link)] with anti-DNP antibody after membrane derivatization (D9656, Sigma-Aldrich, Taufkirchen, Germany). Specific bands were detected by using a chemiluminescence reagent in the ChemiDoc™ Imaging System with ImageLab software (Bio-Rad, Munich, Germany). Band intensities were quantified via densitometric analysis using Image Lab 5.2.1 and Image J software and were normalized to protein content exemplified by Ponceau staining or total unphosphorylated proteins (JNK and AKT phosphorylation).
6
Western Blot Analysis of Cell Signaling Pathways
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Cells were lysed at indicated times p.i. in RIPA buffer supplemented with EDTA-free complete protease inhibitor mixture (Roche, Basel, Switzerland) and phosphatase inhibitors (Cocktail set II, Merck, Darmstadt, Germany). Total protein concentration was determined using a bicinchoninic acid assay. Protein samples were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (BioRad, Hercules, CA, USA). Blots were blocked with 5% milk and incubated with different primary antibodies overnight, followed by incubation with a secondary antibody conjugated with horse-radish peroxidase (Agilent Dako, Santa Clara, CA, USA). Proteins were detected using a BM Chemiluminescence Blotting Substrate (POD) (Roche) or SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) on X-ray films.
Primary antibodies anti-p44/42 MAPK (Erk1/2), anti-phospho-p44/42 MAPK (Erk 1/2), anti-p38MAPK, anti-phospho-p-38 MAPK, anti-JNK2, anti-phospho-SAPK/JNK, anti-IκBα, and anti-phospho-IκBα were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-IL-1β and anti-phospho-IL-1β were purchased from Abcam (Cambridge, UK); anti-Actin and anti-GAPDH for loading controls were purchased from Sigma-Aldrich.
Primary antibodies anti-p44/42 MAPK (Erk1/2), anti-phospho-p44/42 MAPK (Erk 1/2), anti-p38MAPK, anti-phospho-p-38 MAPK, anti-JNK2, anti-phospho-SAPK/JNK, anti-IκBα, and anti-phospho-IκBα were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-IL-1β and anti-phospho-IL-1β were purchased from Abcam (Cambridge, UK); anti-Actin and anti-GAPDH for loading controls were purchased from Sigma-Aldrich.
7
Western Blot Analysis of Signaling Proteins
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MSCs were lysed with RIPA buffer (MilliporeSigma) per the manufacturer’s instructions, and proteins were denatured in SDS sample buffer with 9% β-mercaptoethanol at 100°C for 5 minutes. The following antibodies were used (all purchased from Cell Signaling Technology): anti-SMAD2 (catalog 5339), anti–phospho-SMAD2 (catalog 3104), anti–c-JUN (catalog 9165), anti–phospho-c-JUN (catalog 3270), anti-ERK1/2 (catalog 4695), anti–phospho-ERK1/2 (catalog 4370), anti-JNK1 (catalog 3708), anti-JNK2 (catalog 4672), and anti-vinculin (catalog 13901).
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