rabbit anti-mouse antibodies against jnk  (ABclonal Biotechnology)

Journal: Molecular Biology of the Cell

Article Title: Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-β

doi: 10.1091/mbc.E13-09-0537

Figure Lengend Snippet: Dab2 reduces JNK activation by TGF-β. (A) Knockdown of Dab2 enhances TGF-β–mediated JNK/c-Jun activation. ES-2 cells were transfected with siRNA (nontargeting or directed against Dab2). At 48 h posttransfection, the cells were serum starved (30 min), stimulated (or not) with 100 pM TGF-β1 (30 or 120 min), and analyzed (see Materials and Methods ) by immunoblotting for p-JNK, t-JNK pc-Jun, tc-Jun, pSmad2/3, tSmad2/3, and β-actin. (B) Stable expression of Dab2 abrogates JNK activation. ES-2 and ES-2-Dab2 cells were activated by TGF-β1 (for 30 or 60 min) and analyzed by Western blotting as in A. (C) Quantification of JNK phosphorylation in cells expressing different Dab2 levels (as shown in A and B). Cells transfected with siDab2 demonstrate significantly higher level of pJNK/tJNK ratio compared with ES-2 or ES-2-Dab2 cells. Each bar is the mean ± SEM of three independent experiments (** p < 0.01). (D) Transient overexpression of Dab2 prevents TGF-β stimulation of JNK/c-Jun. Caov3 cells were transfected with GFP (control) or GFP-Dab2. At 24 h posttransfection, the cells were serum starved (60 min), stimulated (or not) with 100 pM TGF-β1 (30 or 60 min), and analyzed as described. All blots shown are of representative experiments ( n = 3 in each case).

Article Snippet: Rabbit IgG against total JNK (α-tJNK), phospho-JNK (α-pJNK), phospho-c-Jun (pc-Jun), and mouse anti–total c-Jun (α-tc-Jun) were from Cell Signaling (Beverly, MA).

Techniques: Activation Assay, Knockdown, Transfection, Western Blot, Expressing, Phospho-proteomics, Over Expression, Control

Journal: Molecular Biology of the Cell

Article Title: Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-β

doi: 10.1091/mbc.E13-09-0537

Figure Lengend Snippet: JNK phosphorylation does not require TβRI kinase activity. ES-2 cells were transfected with siRNA (siControl or siDab2). At 48 h posttransfection, the cells were left untreated or treated with SB431542 (10 μM, 1 h), stimulated (or not) with 100 pM TGF-β1 (30 min), and analyzed (see Materials and Methods ) by immunoblotting for pJNK, tJNK, pc-Jun, tc-Jun, pSmad2/3, and tSmad2/3. The blot shown is of a representative experiment ( n = 3). No significant differences in the fold increase of pJNK/tJNK or of pc-Jun/tc-Jun after TGF-β stimulation were observed between untreated and SB431542-treated cells.

Article Snippet: Rabbit IgG against total JNK (α-tJNK), phospho-JNK (α-pJNK), phospho-c-Jun (pc-Jun), and mouse anti–total c-Jun (α-tc-Jun) were from Cell Signaling (Beverly, MA).

Techniques: Phospho-proteomics, Activity Assay, Transfection, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-β

doi: 10.1091/mbc.E13-09-0537

Figure Lengend Snippet: TGF-β-stimulated JNK phosphorylation is cholesterol dependent. ES-2 cells were transfected with siRNA (siControl or siDab2). At 36 h posttransfection, cells were subjected (or not) to metabolic cholesterol depletion (CD; 12 h). Alternatively, cells at 48 h posttransfection were left untreated or treated with nystatin (25 μg/ml, 2 h). In both experimental conditions, cells were then stimulated (or not) with 100 pM TGF-β1 (30 min), and analyzed (see Materials and Methods ) by immunoblotting for Dab2, pJNK, tJNK, pSmad2/3, and tSmad2/3. The blot shown is of a representative experiment ( n = 3). Although the pJNK/tJNK ratio significantly increased in untreated siDab2 cells after stimulation with TGF-β ( p < 0.05), no significant increase was observed in these cells after treatment with nystatin or cholesterol depletion.

Article Snippet: Rabbit IgG against total JNK (α-tJNK), phospho-JNK (α-pJNK), phospho-c-Jun (pc-Jun), and mouse anti–total c-Jun (α-tc-Jun) were from Cell Signaling (Beverly, MA).

Techniques: Phospho-proteomics, Transfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Adaptor Protein Nck1 Mediates Endothelin A Receptor-regulated Cell Migration through the Cdc42-dependent c-Jun N-terminal Kinase Pathway

doi: 10.1074/jbc.m402767200

Figure Lengend Snippet: FIG. 3. Nck1 is involved in ETA receptor-induced JNK activa- tion. JNK activity (A–C, E, and F) and ERK activity (D) were measured as described under ‘‘Experimental Procedures.’’ Cells co-transfected with the ETA receptor (A–C) and dominant negative mutants of Nck1 (A), Grb2 (B), or CrkII (C) were treated with ET-1 (100 nM, 20 min). Endogenous JNK was immunoprecipitated with anti-JNK antibody and blotted with anti-phosphorylated JNK antibody or anti-JNK antibody. The levels of JNK phosphorylation were quantified and normalized against the total immunoprecipitated JNK levels. D–F, cells transfected with the dominant negative mutants of adaptor proteins were treated with EGF (10 ng/ml, 10 min). The cell lysates were blotted with an anti-phosphorylated ERK or anti-ERK antibody. The levels of ERK or JNK phosphorylation were quantified and normalized against the total immunoprecipitated ERK or JNK levels. Expression of the ETA recep- tor (A–C) and the dominant negative mutants of adaptor proteins (A–F) is shown.

Article Snippet: Materials—Mouse monoclonal antibodies against active phosphorylated JNK (Thr183/Tyr185), active phosphorylated ERK (Thr202/Tyr204), and rabbit polyclonal antibodies against JNK and ERK were purchased from Cell Signaling Technology, Inc (Beverly, MA).

Techniques: Activity Assay, Transfection, Dominant Negative Mutation, Immunoprecipitation, Phospho-proteomics, Expressing

Journal: Journal of Biological Chemistry

Article Title: The Adaptor Protein Nck1 Mediates Endothelin A Receptor-regulated Cell Migration through the Cdc42-dependent c-Jun N-terminal Kinase Pathway

doi: 10.1074/jbc.m402767200

Figure Lengend Snippet: FIG. 6. Effects of siNck1 on the ETA receptor-induced inhibi- tion of cell migration and Cdc42/JNK activation. A, sequence of the synthetic siRNA duplex targeting Nck1 (siNck1–1 or siNck1–2). The 2-nucleotide 3 overhang of 2-deoxythymidine is indicated as TT. The indicated amount of siRNAs was transfected using LipofectAMINE 2000 into the cells. The cell lysates were immunoblotted with anti-Nck1 or anti-tubulin antibodies. B, cells were transfected with siNck1–1 or siGFP using LipofectAMINE 2000. The phosphorylated JNK was de- tected after the addition of ET-1. C, cells were transfected with siNck1–1 or siGFP. After incubation with ET-1, the migrating cells in the Boyden chambers were stained and counted. Data were evaluated using the Student’s t test. Asterisks indicate p 0.01.

Article Snippet: Materials—Mouse monoclonal antibodies against active phosphorylated JNK (Thr183/Tyr185), active phosphorylated ERK (Thr202/Tyr204), and rabbit polyclonal antibodies against JNK and ERK were purchased from Cell Signaling Technology, Inc (Beverly, MA).

Techniques: Migration, Activation Assay, Sequencing, Transfection, Incubation, Staining

Journal: Journal of Biological Chemistry

Article Title: The Adaptor Protein Nck1 Mediates Endothelin A Receptor-regulated Cell Migration through the Cdc42-dependent c-Jun N-terminal Kinase Pathway

doi: 10.1074/jbc.m402767200

Figure Lengend Snippet: FIG. 7. The Src kinase-induced inhibition of cell migration and activation of JNK involves Nck1. Cells were co-transfected with CA-Src and dominant negative mutants of Nck1. After incubation for 5 h, the cells attached to the filters were stained and counted (A). The phosphorylated JNK was analyzed 20 min after stimulation with ET-1 (B). Expression of CA-Src and the dominant negative mutants of Nck1 is shown.

Article Snippet: Materials—Mouse monoclonal antibodies against active phosphorylated JNK (Thr183/Tyr185), active phosphorylated ERK (Thr202/Tyr204), and rabbit polyclonal antibodies against JNK and ERK were purchased from Cell Signaling Technology, Inc (Beverly, MA).

Techniques: Inhibition, Migration, Activation Assay, Transfection, Dominant Negative Mutation, Incubation, Staining, Expressing