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: Bioconjugate chemistry

Article Title: Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

doi: 10.1021/acs.bioconjchem.5b00613

Figure Lengend Snippet: Fig. 6. Detection of proteins in cell lysates. A panel of 24-plex antibody binders was used to detect their respective target proteins in MCF7, K562, A549, and A431 cell lysates. The cell lysates were diluted to different concentrations and reacted with a mixture of antibody binders. Only selected examples of protein targets CSTB (A), CASP3 (B), Ki-67(C), and GATA3 (D) are shown. Ct values were shown as the means ± SD. Ct: cycle threshold.

Article Snippet: MCF7, K562, A549, and A431 cell lysates were obtained from Novus (Littleton, CO).

Techniques:

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: ACS Chemical Neuroscience

Article Title: trans -Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

doi: 10.1021/cn300143m

Figure Lengend Snippet: Cytoprotective doses of trans-resveratrol. Effect on percent cell viability in PC12 cells by MTT assay following (1–1000 μM) concentrations of trans-resveratrol for various time intervals (24–96 h). Values are mean ± SEM of three experiments each carried out in triplicate.

Article Snippet: PC12 cell line was provided by the National Centre for Cell Sciences (NCCS), Pune, India.

Techniques: MTT Assay

Journal: ACS Chemical Neuroscience

Article Title: trans -Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

doi: 10.1021/cn300143m

Figure Lengend Snippet: Protective potential of trans-resveratrol (5, 10, and 25 μM) on percent cell viability: (a) MTT assay, (b) NRU assay, and (c) LDH assay in PC12 cells following the 6 h OGD and 24 h reoxygenation. **P < 0.01 when OGD is compared with normoxia control; ##P < 0.01 when treatment groups are compared with OGD. Values are mean ± SEM of three experiments each carried out in triplicate.

Article Snippet: PC12 cell line was provided by the National Centre for Cell Sciences (NCCS), Pune, India.

Techniques: MTT Assay, NRU Assay, Lactate Dehydrogenase Assay, Control

Journal: ACS Chemical Neuroscience

Article Title: trans -Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

doi: 10.1021/cn300143m

Figure Lengend Snippet: Intracellular calcium levels in PC12 cells following OGD of 6 h and reoxygenation of 24 h and effect of 5, 10, and 25 μM concentrations of trans-resveratrol. **P < 0.01 when OGD is compared with normoxia control, #P < 0.05; ##P < 0.01 when treatment groups are compared with OGD. Values are mean ± SEM of three experiments each carried out in triplicate.

Article Snippet: PC12 cell line was provided by the National Centre for Cell Sciences (NCCS), Pune, India.

Techniques: Control

Journal: ACS Chemical Neuroscience

Article Title: trans -Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

doi: 10.1021/cn300143m

Figure Lengend Snippet: (a) Representative microphotographs showing 6 h OGD and 24 h reoxygenation-induced reactive oxygen species (ROS) generation and effect of 5, 10, and 25 μM concentrations of trans-resveratrol in PC12 cells. Images were captured via a Nikon phase contrast fluorescence microscope (model 80i) with an attached 12.7 megapixel Nikon DS-Ri1 digital CCD cool camera. Values are mean ± SEM of three experiments each carried out in triplicate. (b) Fold change in reactive oxygen species generation. Quantification of fluorescence was done using image analysis software Leica Q-win 500, and data expressed in-fold of unexposed control. **P < 0.01 when OGD is compared with normoxia control; ##P < 0.01 when treatment groups are compared with OGD. Values are mean ± SEM of three experiments each carried out in triplicate.

Article Snippet: PC12 cell line was provided by the National Centre for Cell Sciences (NCCS), Pune, India.

Techniques: Fluorescence, Microscopy, Software, Control

Journal: ACS Chemical Neuroscience

Article Title: trans -Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

doi: 10.1021/cn300143m

Figure Lengend Snippet: (a) Superoxide dismutase activity and (b) catalase activity in PC12 cells following OGD of 6 h and reoxygenation of 24 h and effect of 5, 10, and 25 μM concentrations of trans-resveratrol. **P < 0.01 when OGD is compared with normoxia control, #P < 0.05; ##P < 0.01 when treatment groups are compared with OGD. Values are mean ± SEM of three experiments each carried out in triplicate.

Article Snippet: PC12 cell line was provided by the National Centre for Cell Sciences (NCCS), Pune, India.

Techniques: Activity Assay, Control

Journal: ACS Chemical Neuroscience

Article Title: trans -Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

doi: 10.1021/cn300143m

Figure Lengend Snippet: Relative quantification of-fold induction in the expression of (a) HIF-1α, (b) Cavβ 3, (c) TRPM7, (d) Stat3, and (e) hsp-27 using real time PCR in PC12 cells following OGD of 6 h and reoxygenation of 24 h and effect of 25 μM treatment of trans-resveratrol. HPRT was used as internal control to normalize the data. Quantitative real time PCR (RT-PCRq) was performed in triplicate using SYBR Green dye and an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). **P < 0.01 when OGD is compared with normoxia control, #P < 0.05; ##P < 0.01 when treatment groups are compared with OGD. Values are mean ± SEM of three experiments each carried out in triplicate.

Article Snippet: PC12 cell line was provided by the National Centre for Cell Sciences (NCCS), Pune, India.

Techniques: Quantitative Proteomics, Expressing, Real-time Polymerase Chain Reaction, Control, SYBR Green Assay, Sequencing

Journal: ACS Chemical Neuroscience

Article Title: trans -Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

doi: 10.1021/cn300143m

Figure Lengend Snippet: Alterations in the expression of proteins HIF-1α, Cav β3, TRPM7, STAT3, and hsp-27 using Western blot analysis in PC12 cells following OGD of 6 h and reoxygenation of 24 h and effect of 25 μM treatment of trans-resveratrol. HPRT was used as internal control to normalize the data. Quantification was done using a Gel Documentation system (Alpha Innotech) with the help of Alpha Ease FC StandAlone V.4.0 software. **P < 0.01 when OGD is compared with normoxia control, #P < 0.05; ##P < 0.01 when treatment groups are compared with OGD. Values are mean ± SEM of three experiments each carried out in triplicate.

Article Snippet: PC12 cell line was provided by the National Centre for Cell Sciences (NCCS), Pune, India.

Techniques: Expressing, Western Blot, Control, Software