Journal: Oncotarget

Article Title: Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

doi: 10.18632/oncotarget.6508

Figure Lengend Snippet: MCF7 cells were exposed to ethanol (0 or 100 mg/dl) for 10 days, 1 month or 2 months. A. Cell lysates were collected and then equal amount of proteins were immuoprecipitated (IP) with an anti-p38γ MAPK antibody and then immunoblotted (IB) with an antibody directed against pan phosphorylated p38 MAPK (p-p38). B. Proteins were IP with an anti p-p38 antibody and then IB with an anti-p38α antibody. C. MCF7 cells were exposed to ethanol (0, 100, 200 or 400 mg/dl) for indicated times, then proteins were collected and IP with an anti-p-p38 antibody and IB with an anti-p38γ MAPK antibody. D. MCF7 cells were exposed to ethanol (100 mg/dl) for 0.5–12 hours. The expression of phosphorylated p38γ MAPK, total p38γ MAPK and RhoC was determined by immunoblotting. E. Equal amount of proteins were IP with p38γ or p38α, and then IB with either a commercial anti-pan phosphorylated p38 antibody (p-p38) or a specific anti-phosphorylated-p38γ antibody (p-p38γ) (21st Century Biochemical, please see Materials and Methods). F. The same protein samples described on panel A was analyzed with immunoblotting using the specific anti-p-p38γ MAPK antibody. G. The expression of phosphorylated p38γ in MCF7 and SP-MCF7 cells was evaluated using a specific anti-p-p38γ antibody as described above. All experiments were replicated at least three times.

Article Snippet: Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

doi: 10.18632/oncotarget.6508

Figure Lengend Snippet: A. MCF7 cells were exposed to ethanol (0, 100, 200 or 400 mg/dl) for 10 days, 1 month or 2 months, then the expression of RhoC was examined by immunoblotting. B. MCF7 cells stably expressing control shRNA and p38γ shRNA (p38γsh) were exposed to ethanol (0 or 100 mg/dl) for 10 days. After that, cell lysates were IP with an anti-p38γ antibody, and then IB with an anti p-p38 MAPK antibody. The expression of RhoC was examined. C. Cell lysates were IP with an anti-ubiquitin antibody and IB with an anti-RhoC antibody. D. MCF7 cells were exposed to ethanol (0 or 100 mg/dl) for 10 days, and then treated with either control siRNA or RhoC siRNA for 48 hours. After that, cells were assayed for the migration during a 12-hour-period with/without ethanol (100 mg/dl). E. Cell invasion was evaluated. Each data point was the mean ± SEM of three experiments and presented relative to the controls values. * denotes significant difference from the controls. # denotes significant difference from long term EtOH (LT EtOH). δ denotes significant difference from corresponding controls.

Article Snippet: Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing, Western Blot, Stable Transfection, Control, shRNA, Ubiquitin Proteomics, Migration

Journal: Molecular and Cellular Biology

Article Title: p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261

doi: 10.1128/mcb.01105-15

Figure Lengend Snippet: Figure 1. p38β possesses a trans-autophosphorylation activity that does not target 856

Article Snippet: 24 170 hours later cells were transfected with 3.4, 6.8 or 13.6 μl of 10 μM p38β siRNA 171 (Santa Cruz sc-39116) mixed with 10 μl of the Transit X2 transfection reagent 172 (Myrus) according to manufactures instructions.

Techniques: Activity Assay

Journal: Molecular and Cellular Biology

Article Title: p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261

doi: 10.1128/mcb.01105-15

Figure Lengend Snippet: Figure 3. Novel autophosphorylation sites in p38β identified by LC-capillary 888

Article Snippet: 24 170 hours later cells were transfected with 3.4, 6.8 or 13.6 μl of 10 μM p38β siRNA 171 (Santa Cruz sc-39116) mixed with 10 μl of the Transit X2 transfection reagent 172 (Myrus) according to manufactures instructions.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261

doi: 10.1128/mcb.01105-15

Figure Lengend Snippet: Figure 7. p38α and p38β, but not p38γ or p38δ, possess a general trans-isoform 950

Article Snippet: 24 170 hours later cells were transfected with 3.4, 6.8 or 13.6 μl of 10 μM p38β siRNA 171 (Santa Cruz sc-39116) mixed with 10 μl of the Transit X2 transfection reagent 172 (Myrus) according to manufactures instructions.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261

doi: 10.1128/mcb.01105-15

Figure Lengend Snippet: Figure 8. T241 of p38β is phosphorylated in mammalian cells grown in culture. 957

Article Snippet: 24 170 hours later cells were transfected with 3.4, 6.8 or 13.6 μl of 10 μM p38β siRNA 171 (Santa Cruz sc-39116) mixed with 10 μl of the Transit X2 transfection reagent 172 (Myrus) according to manufactures instructions.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Antrodia cinnamomea Galactomannan Elicits Immuno-stimulatory Activity Through Toll-like Receptor 4

doi: 10.7150/ijbs.24564

Figure Lengend Snippet: ACP induces cytokine expression through MAPK. ( A ) J774A.1 macrophages were incubated for 0-60 min with or without ACP (100 µg/mL). The phosphorylation levels of ERK1/2, JNK1/2 and p38 were assayed by Western blot. ( B, C ) J774A.1 macrophages stably transfected with a control shRNA plasmid (sh-SC), a ERK1 shRNA plasmid (sh-ERK1), a JNK1 shRNA plasmid (sh-TLR2), a p38α shRNA plasmid (sh-p38α), and a p38β shRNA plasmid (sh-p38β) were incubated for 24 h with or without ACP (100 μg/mL). The levels of TNF-α ( B ) and IL-6 ( C ) in the culture medium were measured by ELISA. The Western blot results are representative of those obtained in three different experiments. The ELISA data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Article Snippet: All plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): ERK1 shRNA plasmid (sh-ERK1, sc-29308-SH), JNK1 shRNA plasmid (sh-JNK1, sc-29381-SH), p38α shRNA plasmid (sh-p38α, sc-29434-SH), p38β shRNA plasmid (sh-p38β, sc-39117-SH), TLR2 shRNA plasmid (sh-TLR2, sc-40257-SH) and TLR4 shRNA plasmid (sh-TLR4, sc-40261-SH).

Techniques: Expressing, Incubation, Western Blot, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Molecular signaling underlying bulleyaconitine A (BAA)-induced microglial expression of prodynorphin

doi: 10.1038/srep45056

Figure Lengend Snippet: Effects of bulleyaconitine A (BAA) on levels of phosphorylated p38 ( A ), ERK ( B ) and JNK MAPK ( C ), and blockade effects of the specific inhibitors of p38, ERK and JNK on BAA-induced expression of prodynorphin mRNA level. ( D ) in primary cultures of microglia, and spinal mechanical antiallodynia in neuropathic rats ( E ). Primary microglial cells were collected from the spinal cord of 1-day-old neonatal rats. For phosphorylation, 1 μM BAA was added into microglia culture for 1 hour and levels of phosphorylated p38, ERK, JNK and CREB were determined by using Western blot. The representative blots are shown in the upper panels of each panel. For gene expression, the MAPK inhibitors were incubated 30 minutes before BAA treatment and primary microglial cells were collected 2 hours later. Expression of the dynorphin A precursor prodynorphin gene was determined using qRT-PCR and normalized to GAPDH mRNA. For mechanical antiallodynia, neuropathic rats were induced by tight ligation of L5/L6 spinal nerves, and mechanical thresholds were measured in both contralateral and ipsilateral paws. ( F ) Blockade effect of the p38 inhibitor SB203580 on BAA-induced CREB phosphorylation in cultured primary microglia. Data are expressed as means ± SEM (n = 3 in each treatment with two independent repeats in cultured primary microglial cells, and n = 6 in each group of neuropathic rats). a,b P < 0.05 using the unpaired and two-tailed Student’s t -test or two-way repeated measures ANOVA followed by the post-hoc student-Newman-Keuls test, compared with the control and BAA groups, respectively.

Article Snippet: The small interference of double-stranded RNA (siRNA) targeting the cDNA sequence of the rat p38α (siRNA/p38α), p38β (siRNA/p38β), and the nonspecific oligonucleotides were designed and synthesized by Shanghai GenePharma (Shanghai, China).

Techniques: Expressing, Phospho-proteomics, Western Blot, Gene Expression, Incubation, Quantitative RT-PCR, Ligation, Cell Culture, Two Tailed Test, Control

Journal: Scientific Reports

Article Title: Molecular signaling underlying bulleyaconitine A (BAA)-induced microglial expression of prodynorphin

doi: 10.1038/srep45056

Figure Lengend Snippet: Differential effects of siRNA/p38α and siRNA/p38β on expression of p38α ( A , B ) and p38β mRNA and protein ( C , D ), bulleyaconitine A-induced p38 phosphorylation ( E ) and prodynorphin gene expression ( F ) in primary cultures of microglia. Primary microglial cells were collected from the spinal cord of 1-day-old neonatal rats. For the gene knockdown, siRNA/p38α and siRNA/p38β were transfected into primary microglia for 5 hours and microglia were cultured for additional 24 hours after refreshed the culture medium. Expressions of the mRNA of p38α, p38β and prodynorphin and protein of p38α and p38β referred to gapdh were determined using qRT-PCR and Western blot, respectively. For the phosphorylation assay, primary microglial cells were treated with 1 μM BAA for 1 hour and then subjected to Western blot analysis of phosphorylated p38 with an anti-phospho-p38 antibody. The representative blots are shown in the upper panels of each panel. Data are expressed as means ± SEM (n = 3 per group in each treatment with three independent repeats). a,b P < 0.05 using one-way or two-way repeated measures ANOVA followed by the post-hoc student-Newman-Keuls test, compared with the nonspecific oligonucleotide control and BAA groups, respectively.

Article Snippet: The small interference of double-stranded RNA (siRNA) targeting the cDNA sequence of the rat p38α (siRNA/p38α), p38β (siRNA/p38β), and the nonspecific oligonucleotides were designed and synthesized by Shanghai GenePharma (Shanghai, China).

Techniques: Expressing, Phospho-proteomics, Gene Expression, Knockdown, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: Molecular signaling underlying bulleyaconitine A (BAA)-induced microglial expression of prodynorphin

doi: 10.1038/srep45056

Figure Lengend Snippet: Differential effects of multiple-daily intrathecal injection of siRNA/p38α and siRNA/p38β MAPK on baseline pain thresholds ( A ), bulleyaconitine A (BAA)-induced mechanical antiallodynia ( B ), and expression of spinal p38α ( C and D ) and p38β ( E and F ) mRNA and protein in neuropathic rats. Neuropathic rats, induced by tight ligation of L5/L6 spinal nerves, and then received multiple-daily intrathecal injection of the vehicle (DOTAP, 30 μg/day), nonspecific oligos (5 μg/day), siRNA/p38α (5 μg/day) or siRNA/p38β (5 μg/day) for 7 days. The paw withdrawal thresholds were measured once a day using electronic von frey filaments. On the eighth day, a single bolus of saline (10 μL) or BAA (300 ng) was intrathecally injected and mechanical nociceptive behaviors were quantified 1 hour after injection. Spinal lumbar enlargements were immediately obtained after completion of behavioral assessments. Expressions of the mRNA of p38α, p38β and prodynorphin and protein of p38α and p38β referred to gapdh were determined using qRT-PCR and Western blot, respectively. The representative blots are shown in the upper panels of each panel. Data are expressed as means ± SEM (n = 6 in each group). a,b P < 0.05 using one-way or two-way repeated measures ANOVA followed by the post-hoc student-Newman-Keuls test, compared with the control and BAA groups, respectively.

Article Snippet: The small interference of double-stranded RNA (siRNA) targeting the cDNA sequence of the rat p38α (siRNA/p38α), p38β (siRNA/p38β), and the nonspecific oligonucleotides were designed and synthesized by Shanghai GenePharma (Shanghai, China).

Techniques: Injection, Expressing, Ligation, Saline, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: Molecular signaling underlying bulleyaconitine A (BAA)-induced microglial expression of prodynorphin

doi: 10.1038/srep45056

Figure Lengend Snippet: Stimulatory effects of bulleyaconitine A (BAA) on the intracellular cAMP level ( A ) and PKA phosphorylation ( B ), and blockade effects of the specific adenylate cyclase inhibitor DDA and PKA inhibitor H-89 on BAA-stimulated prodynorphin gene expression ( C ) in primary cultures of microglia, and spinal mechanical antiallodynia in neuropathic rats ( D ). Primary microglial cells were collected from the spinal cord of 1-day-old neonatal rats. For the cAMP measurement, these microglia were incubated with 1 μM BAA for 30 minutes and the intracellular cAMP accumulation was assayed using a commercial ELISA kit. For phosphorylation of PKA and p38, 1 μM BAA was incubated with cultured microglia for 1 hour, and subjected to Western blot analysis. The representative blots are shown in the upper panels of each panel. For gene expression, the MAPK activation inhibitors were incubated 30 minutes before BAA treatment, and primary microglial cells were collected 2 hours later. The level of dynorphin A precursor prodynorphin mRNA was determined using qRT-PCR and normalized to GAPDH mRNA. For mechanical antiallodynia, neuropathic rats were induced by tight ligation of L5/L6 spinal nerves, and mechanical thresholds were measured in both contralateral and ipsilateral paws. Blockade effects of DDA ( E ) and H-89 ( F ) on BAA-induced p38 phosphorylation were also assessed. Data are presented as means ± SEM (n = 3 in each treatment with two independent repeats in cultured primary microglial cells, and n = 6 in each group of neuropathic rats). a,b P < 0.05 using the unpaired and two-tailed Student’s t -test or two-way repeated measures ANOVA followed by the post-hoc student-Newman-Keuls test, compared with the control and BAA groups, respectively.

Article Snippet: The small interference of double-stranded RNA (siRNA) targeting the cDNA sequence of the rat p38α (siRNA/p38α), p38β (siRNA/p38β), and the nonspecific oligonucleotides were designed and synthesized by Shanghai GenePharma (Shanghai, China).

Techniques: Phospho-proteomics, Gene Expression, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Activation Assay, Quantitative RT-PCR, Ligation, Two Tailed Test, Control

Journal: Scientific Reports

Article Title: Molecular signaling underlying bulleyaconitine A (BAA)-induced microglial expression of prodynorphin

doi: 10.1038/srep45056

Figure Lengend Snippet: Following activation of the “aconitine receptor”, the Gs-protein, cAMP/PKA, p38β (but not ERK, JNK or p38α) MAPK and CREB (but not NF-κB) signals are successively activated, which mediates prodynorphin expression.

Article Snippet: The small interference of double-stranded RNA (siRNA) targeting the cDNA sequence of the rat p38α (siRNA/p38α), p38β (siRNA/p38β), and the nonspecific oligonucleotides were designed and synthesized by Shanghai GenePharma (Shanghai, China).

Techniques: Activation Assay, Expressing