Journal: The Journal of biological chemistry

Article Title: Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase.

doi: 10.1074/jbc.274.19.13193

Figure Lengend Snippet: FIG. 3. The catalytic activity of B-Rafcat is inhibited by co- transfection of PKAcat in PC12 cells. A, a plasmid encoding B- Rafcat-GST was expressed in PC12 cells in the absence (2PKA) or presence (1PKA) of co-transfected PKAcat. MEK kinase activity of the glutathione-Sepharose-purified proteins was determined as described in Fig. 2. An anti-B-Raf immunoblot analysis of the samples used for the MEK kinase assay indicates that an equal amount of B-Rafcat-GST fusion protein was present in each reaction. B, a histogram summariz- ing the inhibitory effect of PKAcat on B-Rafcat activity in transfected PC12 cells from four independent experiments. The error bar indicates the standard deviation of the mean.

Article Snippet: PC12 B-Raf was immunopurified from NGF-stimulated PC12 cells by incubating 100 mg of total cell lysate protein with 2 mg of anti-B-Raf IgG (Santa Cruz Biotechnology) and 30 ml of a 1:1 slurry of protein G Plus-Agarose (Santa Cruz Biotechnology) at 4 °C for 1 h. The immune complex was washed as described above.

Techniques: Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Purification, Western Blot, Kinase Assay, Standard Deviation

Journal: The Journal of biological chemistry

Article Title: Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase.

doi: 10.1074/jbc.274.19.13193

Figure Lengend Snippet: FIG. 4. Activated B-Raf is not inhibited by PKA in vitro. A, activated B-Raf was immunoprecipitated from NGF-stimulated PC12 cells and incubated in the absence (2PKA) or presence (1PKA) of PKA in vitro. Raf-1 was isolated from immature Xenopus oocytes, activated with purified PKC, and subsequently treated in the presence or absence of PKA. MEK kinase activity of the B-Raf and Raf-1 proteins was measured as described in the legend to Fig. 2. In this experiment, B-Raf was activated by NGF to levels 4-fold higher than the activity of B-Raf in unstimulated PC12 cells. Raf-1 was activated by PKC 12.9-fold higher than the level of Raf-1 in immature Xenopus oocytes. KN-MEK, kinase-negative MEK. B, histograms summarizing the inhibitory effect of PKA on NGF-stimulated B-Raf activity and PKC-stimulated Raf-1 activity from three independent experiments. The error bars indicate the standard errors of the mean.

Article Snippet: PC12 B-Raf was immunopurified from NGF-stimulated PC12 cells by incubating 100 mg of total cell lysate protein with 2 mg of anti-B-Raf IgG (Santa Cruz Biotechnology) and 30 ml of a 1:1 slurry of protein G Plus-Agarose (Santa Cruz Biotechnology) at 4 °C for 1 h. The immune complex was washed as described above.

Techniques: In Vitro, Immunoprecipitation, Incubation, Isolation, Purification, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase.

doi: 10.1074/jbc.274.19.13193

Figure Lengend Snippet: FIG. 5. Full-length B-Raf is activated by co-expressed PKAcat in PC12 cells. Full-length B-Raf-GST was expressed in PC12 cells in the absence (2PKA) or presence (1PKA) of co-transfected PKAcat. A, B-Raf immunoblot analysis of glutathione-Sepharose affinity-purified lysates performed in the absence of bacterially expressed kinase-nega- tive MEK. B, MEK kinase activity of glutathione-Sepharose-purified proteins was measured as described in the legend to Fig. 2. The histo- gram summarizes the stimulatory effect of PKAcat on B-Raf-GST from three independent experiments. The error bar indicates the standard error of the mean.

Article Snippet: PC12 B-Raf was immunopurified from NGF-stimulated PC12 cells by incubating 100 mg of total cell lysate protein with 2 mg of anti-B-Raf IgG (Santa Cruz Biotechnology) and 30 ml of a 1:1 slurry of protein G Plus-Agarose (Santa Cruz Biotechnology) at 4 °C for 1 h. The immune complex was washed as described above.

Techniques: Transfection, Western Blot, Affinity Purification, Activity Assay, Purification

Journal: Cells

Article Title: Melatonin Attenuates Spinal Cord Injury in Mice by Activating the Nrf2/ARE Signaling Pathway to Inhibit the NLRP3 Inflammasome

doi: 10.3390/cells11182809

Figure Lengend Snippet: The oxidative damage scavenging effect of melatonin in neuron cells. ( A ) The survival rates of PC12 cells that received different concentrations of melatonin treatment were detected using a CCK-8 kit (n = 8). ( B ) The ROS produced in PC12 cells that received different treatments were detected by ROS staining (n = 6, scale bar = 100 µm). ( C , D ) The mitochondrial membrane potential in PC12 cells treated with drugs was detected by JC-1 staining (n = 8, scale bar = 100 µm). ( E – G ) The contents of SOD and MDA and the activity of GSH-PX were detected using the corresponding kits in PC12 cells treated with different treatment factors (n = 10). Data are shown as the means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-18 were measured in the PC12 cell culture medium using ELISA kits according to the manufacturer’s instructions (Elabscience, China).

Techniques: CCK-8 Assay, Produced, Staining, Membrane, Activity Assay

Journal: Cells

Article Title: Melatonin Attenuates Spinal Cord Injury in Mice by Activating the Nrf2/ARE Signaling Pathway to Inhibit the NLRP3 Inflammasome

doi: 10.3390/cells11182809

Figure Lengend Snippet: Melatonin suppresses the NLRP3 inflammasome in PC12 cells treated with H2O2 by activating the Nrf2/ARE pathway. ( A – I ) Western blot detection and statistical analysis of NLRP3-inflammasome-related proteins NLRP3, caspase-1, ASC, and IL-1β; and antioxidant-stress-related proteins nuclear Nrf2, HO-1, and NQO-1 in PC12 cells from each group (n = 6). ( J , K ) The levels of IL-1β and IL-18 in the supernatant of PC12 cells were determined by ELISA kits (n = 6). Data are shown as the means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-18 were measured in the PC12 cell culture medium using ELISA kits according to the manufacturer’s instructions (Elabscience, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Melatonin Attenuates Spinal Cord Injury in Mice by Activating the Nrf2/ARE Signaling Pathway to Inhibit the NLRP3 Inflammasome

doi: 10.3390/cells11182809

Figure Lengend Snippet: Immunofluorescence double staining of Nrf2 and NLRP3 in PC12 cells treated with H2O2. Immunofluorescence double staining was performed to test the protein level of Nrf2 ( A , B ) and NLRP3 ( C , D ) in PC12 cells from each group (n = 8, scale bar = 100 µm). Data are shown as the means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-18 were measured in the PC12 cell culture medium using ELISA kits according to the manufacturer’s instructions (Elabscience, China).

Techniques: Immunofluorescence, Double Staining

Journal: Cell Adhesion & Migration

Article Title: Expression of kindlin-3 in melanoma cells impedes cell migration and metastasis

doi: 10.1080/19336918.2016.1243645

Figure Lengend Snippet: Analyses of kindlin-3 expression in M10 human melanoma cell line. (A) qRT-PCR analyses of kindlin-1, −2 and −3 mRNA expression levels in M10 cells and the human chronic myeloid leukemia cell line K562. Values are mean ± SD of technical triplicates. A representative experiment from 3 independent experiments is shown. (B) qRT-PCR analyses of kindlin-3 expression in M10, K562 and the human fibroblast cell line 293T. In (A) and (B), GAPDH and actin were used as internal controls. (C) Immunoblot analyses of kindlin-3 protein expression in M10 cells, K562, 293T and the human breast cancer cell line MDA-MB-231 using the anti-kindlin-3 mAb clone 9. Actin serves as loading control. (D) Immunoblot analyses using the anti-kindlin-3 mAb 3D6.

Article Snippet: Human melanoma cell line M10 was purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI1640 full-medium containing supplements aforementioned.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Adhesion & Migration

Article Title: Expression of kindlin-3 in melanoma cells impedes cell migration and metastasis

doi: 10.1080/19336918.2016.1243645

Figure Lengend Snippet: Analyses of the migratory properties of M10 cells transfected with GFP-kindlin-3. (A) Flow cytometry analyses and sorting of cells transiently transfected with GFP or GFP-kindlin-3 expression plasmid. (B) Sorted cells were subjected to 2D-random migration assays on fibronectin-coated (2 μg/cm2) coverslip-bottom culture dishes. Twenty cells were tracked and migratory profiles were plotted. Representative data from at least 2 independent experiments are shown. X and Y axes are relabeled with larger fonts for clarity.

Article Snippet: Human melanoma cell line M10 was purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI1640 full-medium containing supplements aforementioned.

Techniques: Transfection, Flow Cytometry, Expressing, Plasmid Preparation, Migration