Journal: PLoS ONE

Article Title: Apigenin Induces the Apoptosis and Regulates MAPK Signaling Pathways in Mouse Macrophage ANA-1 Cells

doi: 10.1371/journal.pone.0092007

Figure Lengend Snippet: A–C. Representative western blot results showed phospho-ERK1/2/ERK1/2, phospho-JNK/JNK and phospho-p38/p38 expression in the ANA-1 cells after incubation with apigenin for 48 h. D. The relative expression of proteins compared with the control, *, p <0.05, **, p <0.01, versus the control.

Article Snippet: Mouse monoclonal anti-phospho-ERK1/2, anti-caspase-3, anti-caspase-8, anti-phospho-p38, rabbit monoclonal anti-ERK1/2, anti-p38, mouse monoclonal anti-β-actin, rabbit/goat anti-mouse IgG and goat anti-rabbit IgG antibodies were purchased from Santa Cruz Biotechnology (USA).

Techniques: Western Blot, Expressing, Incubation

Journal: BMC Cancer

Article Title: SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro

doi: 10.1186/1471-2407-8-150

Figure Lengend Snippet: PYK2 expression, Tyr402 and ERK1/2 phosphorylations, as well as cell migration in HBE and A549 cells. (a) PYK2 expression, Tyr402 and ERK1/2 phosphorylations in HBE and A549 cells by western blot. (b) Comparisons of migratory abilities between HBE and A549 cells by Transwell assay. The data are representative of three individual experiments.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-myc tag (Applygen, China), rabbit polyclonal anti-SOCS3, anti-PYK2, anti-p-Tyr40, anti-β-actin, and mouse monoclonal anti-p-ERK1/2 antibodies (all from Santa Cruz).

Techniques: Expressing, Migration, Western Blot, Transwell Assay

Journal: BMC Cancer

Article Title: SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro

doi: 10.1186/1471-2407-8-150

Figure Lengend Snippet: Up-regulation of SOCS3 by 5-aza-2'-deoxycytidine treatment inhibited cell migration and associated PYK2 expression, Tyr402 and ERK1/2 phosphorylations. (a) Decreased PYK2 expression, Tyr402 and ERK1/2 activations and migration of A549 cells were observed by SOCS3 up-regulation. A549 cells were treated with 5-aza-2'-deoxycytidine for 6 d, and cell lysates were analysed by western blot using the indicated antibodies. Elevated PYK2 expression, Tyr402 and ERK1/2 phosphorylations were found by the pretreatment of β-lactacystin. (b) PYK2 mRNA levels remained invariable regardless of treatment or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration was suppressed by SOCS3 restoration, and the β-lactacystin pretreatment facilitated cell migration to some extent. The values are means of three replicates.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-myc tag (Applygen, China), rabbit polyclonal anti-SOCS3, anti-PYK2, anti-p-Tyr40, anti-β-actin, and mouse monoclonal anti-p-ERK1/2 antibodies (all from Santa Cruz).

Techniques: Migration, Expressing, Western Blot, Amplification

Journal: BMC Cancer

Article Title: SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro

doi: 10.1186/1471-2407-8-150

Figure Lengend Snippet: Effects of SOCS3-KIR mutant on PYK2 expression, Tyr402 and ERK1/2 activations and migration of A549 cells . (a) Down-regulations of PYK2 expression, Tyr402 and ERK1/2 phosphorylations by SOCS3-KIR mutant. A549 cells were transfected with either vector or the SOCS3-KIR mutant for 48 h, and cell lysates were subjected to western blot using the indicated antibodies. Cells pretreated with β-lactacystin showed that PYK2 expression, Tyr402 and ERK1/2 phosphorylations were restored to some extent. (b) PYK2 mRNA levels were unaffected regardless of transfection or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration inhibited by the SOCS3-KIR mutant was statistically significant, and β-lactacystin pretreatment restored cell migration to some degree. The values are means of three replicates.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-myc tag (Applygen, China), rabbit polyclonal anti-SOCS3, anti-PYK2, anti-p-Tyr40, anti-β-actin, and mouse monoclonal anti-p-ERK1/2 antibodies (all from Santa Cruz).

Techniques: Mutagenesis, Expressing, Migration, Transfection, Plasmid Preparation, Western Blot, Amplification

Journal: BMC Cancer

Article Title: SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro

doi: 10.1186/1471-2407-8-150

Figure Lengend Snippet: Effects of SOCS-box mutant on PYK2 expression, Tyr402 and ERK1/2 activations and cell migration . (a) Decreased PYK2 and ERK1/2 phosphorylations by SOCS-box mutant with the same PYK2 expression. A549 cells were vector- or the SOCS-box mutant-transfected for 48 h, and cell lysates were analysed by western blot using the indicated antibodies. (b) PYK2 mRNA levels were unaffected regardless of transfection or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration suppressed by the SOCS-box mutant was not statistically significant. The values are means of three replicates.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-myc tag (Applygen, China), rabbit polyclonal anti-SOCS3, anti-PYK2, anti-p-Tyr40, anti-β-actin, and mouse monoclonal anti-p-ERK1/2 antibodies (all from Santa Cruz).

Techniques: Mutagenesis, Expressing, Migration, Plasmid Preparation, Transfection, Western Blot, Amplification

Journal: PLoS ONE

Article Title: Prevention of Wear Particle-Induced Osteolysis by a Novel V-ATPase Inhibitor Saliphenylhalamide through Inhibition of Osteoclast Bone Resorption

doi: 10.1371/journal.pone.0034132

Figure Lengend Snippet: ( A ) The effect of saliPhe and bafilomycin on RANKL-induced IκBα phosphorylation and degradation. M-CSF-dependent BMMs pretreated with or without saliPhe (80 nM) or bafilomycin (1.25 nM) were stimulated with 100 ng/ml RANKL for the indicated duration. Cells were lysed and analysed by western blot using anti-p-IκBα and anti-IκBα antibody. Antibody against β-actin was used as loading control. ( B ) The effect of saliPhe and bafilomycin on NF-κB activation. RAW264.7 cells stably transfected with the p-NF-κB-TA-Luc luciferase reporter gene were pretreated with saliPhe (80 nM) or bafilomycin (1.25 nM) prior to stimulation with RANKL (100 ng/ml) for 8 hrs. Cell lysates were harvested and luciferase activity was measured. Relative luciferase response was plotted. Data representative of 3 independent experiments performed in triplicates (mean ± SEM; *P<0.05, **P<0.01 against control). ( C ) The effect of saliPhe and bafilomycin on ERK1/2 signalling pathway. BMMs pretreated with or without saliPhe (80 nM) or bafilomycin (1.25 nM) were stimulated by RANKL for the indicated duration. The expression of ERK1/2 and phosphorylated ERK1/2 were probed using corresponding antibodies. ( D ) The effect of saliPhe and bafilomycin on TRAF6 and NFATc1 induction. M-CSF-dependent BMMs pretreated with or without saliPhe (80 nM) or bafilomycin A1 (1.25 nM) was stimulated with 100 ng/ml RANKL for the indicated duration. Cells were lysed and analysed by western blot using anti-TRAF6 and anti-NFATc1 antibody. Antibody against β-actin was used as loading control.

Article Snippet: Antibodies used include mouse monoclonal anti-β-actin (JLA20) (DSHB University of Iowa), rabbit polyclonal anti-IκBα (Santa Cruz), mouse monoclonal anti-p-IκBα (Santa Cruz), rabbit polyclonal anti-ERK1/2 (Promega), mouse monoclonal anti-p-ERK1/2 (Santa Cruz), rabbit polyclonal anti-TRAF6 (Santa cruz) and mouse monoclonal anti-NFATc1 (BD Pharmingen).

Techniques: Western Blot, Activation Assay, Stable Transfection, Transfection, Luciferase, Activity Assay, Expressing