Journal: Molecular Neurodegeneration

Article Title: Increased 90-kDa ribosomal S6 kinase (Rsk) activity is protective against mutant huntingtin toxicity

doi: 10.1186/1750-1326-6-74

Figure Lengend Snippet: Inhibition of Rsk induces a reduction of pBAD and pSRF levels in STHdh Q111/Q111 cells . Lysates from STHdh Q7/Q7 and STHdh Q111/Q111 cells treated with or without BI-D1870 (0.1 μM; BI) were subjected to western blot to analyze pBAD (Ser-112)/Bad (A) and pSRF (Ser-103)/SRF (B) and tubulin protein levels. Results are expressed as the ratio between phospho and total Bad (A) and SRF (B) levels. Data are the mean ± SEM of four independent experiments performed in duplicate. Representative immunoblots are presented. Results are expressed in percentage respect to STHdh Q7/Q7 cells. Data were analyzed using two-way ANOVA followed by Bonferroni's post hoc test. *p < 0.05 and **p < 0.01 as compared with control STHdh Q7/Q7 cells and +++ p < 0.001 as compared with control STHdh Q111/Q111 cells.

Article Snippet: The following primary antibodies were used: anti-Rsk1, anti-Rsk2, anti-phospho-Rsk (Ser-221) and anti-phospho-Rsk (Ser-380) (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-htt monoclonal 2166 (1:1000; Millipore Bioscience Research reagents, Temecula, CA), anti-phospho-SRF (Ser-103), anti-SRF, anti-phospho-Bad (Ser-112) and anti-Bad (all 1:1000; Cell Signaling Technology, Beverly, MA), and anti-HA (1:1000; Sigma-Aldrich, Saint Louis, MO).

Techniques: Inhibition, Western Blot

Journal: BMC Complementary and Alternative Medicine

Article Title: Electroacupuncture-like stimulation at Baihui and Dazhui acupoints exerts neuroprotective effects through activation of the brain-derived neurotrophic factor-mediated MEK1/2/ERK1/2/p90RSK/bad signaling pathway in mild transient focal cerebral ischemia in rats

doi: 10.1186/1472-6882-14-92

Figure Lengend Snippet: Effects of EA at acupoints on cytosolic expression of pMEK1/2, pERK1/2, pp90RSK, and pBad. (A) Representative western blot images showed the cytosolic expression of pMEK1/2, pERK1/2, pp90RSK, and pBad in the ischemic cortex in the sham, model, EA, non-acup, and U0126 + EA groups after 3 d of reperfusion. Actin was used as an internal control. The relative cytosolic expression of (B) pMEK1/2, (C) pERK1/2, (D) pp90RSK, and (E) pBad (n = 4) was assessed in the ischemic cortex in the sham, model, EA, non-acup, and U0126 + EA groups. Data are presented as mean ± SD. # P < 0.05 compared with the model group.

Article Snippet: They were then incubated with a rabbit anti-pMEK1/2 (1:1000 dilution, #2338 Cell Signaling Technology), rabbit anti-pERK1/2 (1:1000 dilution, #4376 Cell Signaling Technology), rabbit anti-pp90RSK (1:1000 dilution, #9344 Cell Signaling Technology), or rabbit anti-phospho-Bad (pBad) (1:1000 dilution, #9291 Cell Signaling Technology) antibody overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Unfertilized Xenopus Eggs Die by Bad-Dependent Apoptosis under the Control of Cdk1 and JNK

doi: 10.1371/journal.pone.0023672

Figure Lengend Snippet: (A) Oocytes were microinjected with a mRNA encoding Bad-GFP and one hour later incubated in the presence or the absence of the JNK inhibitor, SP600125, or microinjected with p21 Cip1 protein (Cip). They were then stimulated with progesterone (time 0). Egg proteins were analysed at the indicated times by immunoblot using antibodies against Bad, Ser128 (pS128), Ser112 (pS112) or Ser136 (pS136)-phosphorylated forms of Bad, and the active phosphorylated form of JNK (p-JNK). These data are representative experiments repeated on oocytes and eggs from at least three different females. (B) Regulation of apoptosis in unfertilized eggs. In the ovary, prophase oocytes are protected from apoptosis by an inhibited form of Bad phosphorylated at Ser112 and Ser136. At time of ovulation, the oocyte completes meiotic maturation. Bad becomes phosphorylated on Ser128 under the control of Cdk1 and JNK. During aging, the ovulated egg progressively accumulates increasing amounts of the Ser128 phosphorylated form of Bad that can ultimately trigger the death execution, unless fertilization occurs.

Article Snippet: Samples of 50 µg of proteins (equivalent to 0.25 to 2 oocytes or eggs) in Laemmli buffer were electrophoresed on 10% or 15% SDS-PAGE (Amresco Inc., USA), transferred to nitrocellulose filters (Schleicher and Schuell) using a semi-dry blotting system (Millipore) and immunoblotted using the following antibodies: anti- Xenopus cyclin B2 , anti-phosphorylated ERK 1/2 (New England Biolabs), anti-ERK1 and anti- Xenopus Mos (Santa Cruz Biotechnologies), anti-caspase 9 , anti-Cyt c, anti-Bax, anti-p150Glued (BD Bioscience, USA), anti-phosphorylated histone H2B (Upstate Biotechnology Inc., USA), anti-Ser128 phosphorylated Bad (Abcam, France), anti-caspase 3, anti-Ser10 phosphorylated histone H3, anti-phosphorylated JNK, anti-Bad, anti-Ser112 phosphorylated Bad and anti-Ser136 phosphorylated Bad (Cell Signaling, Danvers, USA).

Techniques: Incubation, Western Blot

Journal: PLoS ONE

Article Title: A Rac1/Cdc42 GTPase-Specific Small Molecule Inhibitor Suppresses Growth of Primary Human Prostate Cancer Xenografts and Prolongs Survival in Mice

doi: 10.1371/journal.pone.0074924

Figure Lengend Snippet: Analysis of PAK, AKT and BAD-phosphorylation in EGF-stimulated 22Rv1 prostate cancer cells following AZA1 treatment. Representative Western blot images and quantification of immunoblots stained with phospho-PAK1/2 (pPAK), phospho-AKT (pAKT) and phospho-BAD (pBAD) antibodies before and after treatment with 2, 5 and 10 µM AZA1 for 24 h. Rac1/Cdc42 blockade reduces phosphorylation of PAK1, AKT and BAD in 22Rv1 prostate cancer cells compared to controls (means of 3 independent experiments). *, significantly different from unstimulated and untreated controls; + , significantly different from EGF-stimulated control; ‡ , significantly different from unstimulated, untreated control and EGF-stimulated control. SP, specific protein; LC, loading control.

Article Snippet: The blots were probed with antibodies against phospho-PAK1 (pS144)/PAK2 (pS141) (Cell Signaling Technology, Danvers, MA), phospho-AKT (anti-phospho-AKT pT308 from BD Biosciences) and phospho-BAD (anti-phospho-BAD Ser112 from Cell Signaling Technology) before incubation with horseradish peroxidase–conjugated secondary antibodies (Amersham Pharmacia Biotech).

Techniques: Western Blot, Staining

Journal: PLoS ONE

Article Title: A Rac1/Cdc42 GTPase-Specific Small Molecule Inhibitor Suppresses Growth of Primary Human Prostate Cancer Xenografts and Prolongs Survival in Mice

doi: 10.1371/journal.pone.0074924

Figure Lengend Snippet: AZA1 inhibits activation of Rac1 and Cdc42 GTPases, shifting the balance towards cell growth inhibition and apoptosis. These effects are exerted by different mechanisms. AZA1 inhibits the PAK pathway via the cell cycle regulator Cyclin D1 and suppresses PAK and AKT activation leading to reduced BAD phosphorylation to induce pro-apoptotic activity. In addition, suppression of Rac1 and Cdc42 suppresses cancer cell migration and invasion by affecting actin polymerization and subsequent inhibition of lamellipodia and filopodia formation.

Article Snippet: The blots were probed with antibodies against phospho-PAK1 (pS144)/PAK2 (pS141) (Cell Signaling Technology, Danvers, MA), phospho-AKT (anti-phospho-AKT pT308 from BD Biosciences) and phospho-BAD (anti-phospho-BAD Ser112 from Cell Signaling Technology) before incubation with horseradish peroxidase–conjugated secondary antibodies (Amersham Pharmacia Biotech).

Techniques: Activation Assay, Inhibition, Activity Assay, Migration