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: Neural Regeneration Research

Article Title: Srgap2 suppression ameliorates retinal ganglion cell degeneration in mice

doi: 10.4103/1673-5374.369122

Figure Lengend Snippet: Downstream signaling pathways after inhibition of Srgap2 . (A) Detection of SRGAP2 (green, Alexa Fluor 488) expression in PC12 cells by immunofluorescence staining. n = 3 independent cell culture preparations. Scale bars: 50 μm. (B) SRGAP2 protein level was decreased after siRNA transfection and Rac1 and Cdc42 were increased after inhibiting Srgap2. (C) Quantification of B. n = 3 independent cell culture preparations. (D) mTOR signaling pathway was activated after the downregulation of Srgap2. (E) Quantification of D. n = 3 independent cell culture preparations. (F, G) ONC induced activation of the mTOR signaling pathway 7 days after ONC. Srgap2 +/– mice showed increased mTOR activation compared with WT. n = 3 independent animals in each group. Data (normalized by WT-ctrl group) are presented as mean ± SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two-tailed Student’s t -test). Cdc42: Cell division cycle 42; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; mTOR: mammalian target of rapamycin; ONC: optic nerve crush; Rac1: Ras-related C3 botulinum toxin substrate 1; siRNA: small interfering RNA; Srgap2: Slit-Robo GTPase activating protein 2; WT: wild type.

Article Snippet: PC12, one of the most commonly used cell lines in neuroscience research (You et al., 2022), was purchased from CHI Scientific (Jiangyin, China; RRID: CVCL_0481).

Techniques: Protein-Protein interactions, Inhibition, Expressing, Immunofluorescence, Staining, Cell Culture, Transfection, Activation Assay, Two Tailed Test, Control, Small Interfering RNA