Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit p44/42 MAPK (Erk1/2) Antibody , Cell Signaling , Cat#9102; 9102S RRID: AB_330744 , 1:4000.

Techniques: Western Blot, Immunostaining, Immunoprecipitation, Protease Inhibitor, Software, Imaging, Slice Preparation, Incubation

Journal: eLife

Article Title: Impairment of cocaine-mediated behaviours in mice by clinically relevant Ras-ERK inhibitors

doi: 10.7554/eLife.17111

Figure Lengend Snippet: ( A–F ) Dose response curves of RB1 and RB3 for ERK, Ac-H3 and S6 activation. 200 μ m thick striatal slices were freshly prepared from 2-month old mice and transferred into a perfusion chamber for 1 hr at 32°C. Slices were pre-treated with either different doses of peptides (RB1 and RB3) or scrambled controls (scramble RB1 or scramble RB3). After 1 hr, one group of slices for each condition was fixed in PFA 4% for 15 min, while another group was stimulated with glutamate 100 μ M for 10 min prior to fixation. 18 μ m cryo-sections were processed for immunohistochemistry with anti-phospho p44/p42 MAP kinase (Thr202/Tyr204) or for immunofluorescence co-labelling the slices with anti-phospho (Ser10) - acetyl (Lys14) H3 or anti-phospho S6 (Ser235/236) and the neuronal marker NeuN. Neuronal quantification was performed with ImageJ software by counting the number of phospho-ERK positive cells and the number of phospho-Ac-H3 or phospho-S6 among NeuN positive neurons in each slice. The level of activation is expressed on the Y-axis as arbitrary units (AU). Doses are reported in a logarithmic scale (Log10) on the X-axis. The IC50 was calculated for each specific response using GraphPad Prism software. DOI: http://dx.doi.org/10.7554/eLife.17111.003

Article Snippet: 1 hr after blocking in 5% normal goat serum and 0.1% Triton X-100 solution, slices were incubated overnight at 4°C with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) (1:200, Cell Signaling Technology Cat# 4370L, RRID: AB_2297462 ).

Techniques: Activation Assay, Immunohistochemistry, Immunofluorescence, Marker, Software

Journal: eLife

Article Title: Impairment of cocaine-mediated behaviours in mice by clinically relevant Ras-ERK inhibitors

doi: 10.7554/eLife.17111

Figure Lengend Snippet: RB1 (20 mg/kg), RB3 (20 mg/kg), or the scrambled peptides (Scr) were administered to wild-type mice 1 hr before an acute cocaine (25 mg/kg) or saline injection. After 5 min mice were perfused. Immunohistochemistry was performed with anti-phospho p44/42 MAP kinase (Thr202/Tyr204, scale bar 30 μ m). Quantification of phospho-ERK positive cells shows a significant inhibitory effect of RB1 and RB3 on cocaine-induced ERK activation in the ventral striatum. Two-way ANOVA: effect of RB1 F 1,14 = 18.73, p<0.001, effect of cocaine F 1,14 = 111.87, p<0.0001, effect of interaction F 1,14 = 23.95, p<0.001; Bonferroni’s post-hoc, Scr RB1 saline (n=5) vs Scr RB1 cocaine (n=3) p<0.001, Scr RB1 cocaine (n=3) vs RB1 cocaine (n=5) p<0.001. Two-way ANOVA: effect of RB3 F 1,14 = 6.26, p<0.05, effect of cocaine F 1,14 = 52.11, p<0.0001, effect of interaction F 1,14 = 16.06, p<0.01; Bonferroni’s post-hoc, Scr RB3 saline (n=5) vs Scr RB3 cocaine (n=3) p<0.001, Scr RB3 cocaine (n=3) vs RB3 cocaine (n=5) p<0.01. **p<0.01, ***p<0.001. Data are shown as mean with SEM. DOI: http://dx.doi.org/10.7554/eLife.17111.005

Article Snippet: 1 hr after blocking in 5% normal goat serum and 0.1% Triton X-100 solution, slices were incubated overnight at 4°C with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) (1:200, Cell Signaling Technology Cat# 4370L, RRID: AB_2297462 ).

Techniques: Saline, Injection, Immunohistochemistry, Activation Assay

Journal: eLife

Article Title: Impairment of cocaine-mediated behaviours in mice by clinically relevant Ras-ERK inhibitors

doi: 10.7554/eLife.17111

Figure Lengend Snippet: 200 μ m thick striatal slices obtained from 2-month old mice were transferred into a perfusion chamber for 1 hr at 32°C in the presence of different doses of PD325901 or vehicle. After 1 hr, one group of slices for each condition was fixed in PFA 4% for 15 min, while another group was stimulated with glutamate 100 μ M for 10 min prior to fixation. 18 μ m cryo-sections were processed for immunohistochemistry with anti-phospho p44/p42 MAP kinase (Thr202/Tyr204). ERK activation is determined by counting the number of phospho-ERK positive neurons in each slice. The level of activation is expressed on the Y-axis as arbitrary units (AU). Doses are reported in logarithmic scale (Log10) on the X-axis. The IC50was calculated for the specific response using GraphPad Prism software. As shown in the graph, PD325901 is effective in reducing glutamate-induced ERK activation with an IC50 of 1.15 nM. Scale bar 100 µm. DOI: http://dx.doi.org/10.7554/eLife.17111.009

Article Snippet: 1 hr after blocking in 5% normal goat serum and 0.1% Triton X-100 solution, slices were incubated overnight at 4°C with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) (1:200, Cell Signaling Technology Cat# 4370L, RRID: AB_2297462 ).

Techniques: Immunohistochemistry, Activation Assay, Software

Journal: eLife

Article Title: Impairment of cocaine-mediated behaviours in mice by clinically relevant Ras-ERK inhibitors

doi: 10.7554/eLife.17111

Figure Lengend Snippet: Different doses of PD325901 were administered i.p. to wild-type mice 1 hr before the saline or cocaine (25 mg/kg) injection. After 5 min mice were perfused. Immunohistochemistry was performed with anti-phospho p44/42 MAP kinase (Thr202/Tyr204, lower panel, scale bar 30 μ m). Quantification of phospho-ERK positive cells in the dorsal striatum (upper panel, mean with SEM) shows that PD325901 inhibited ERK in a dose-dependent manner. Two-way ANOVA, effect of PD325901 F 7,78 = 2.43, p<0.05, effect of cocaine F 1,78 = 4.81, p<0.05, effect of interaction F 7,78 = 3.47, p<0.01; Bonferroni’s post-hoc, vehicle saline (n=9) vs vehicle cocaine (n=15) p<0.0001, vehicle cocaine (n=15) vs PD325901 0.25 mg/kg (n=4) p>0.05, vehicle cocaine (n=15) vs PD325901 0.5 mg/kg (n=5) p<0.05, vehicle cocaine (n=15) vs PD325901 1 mg/kg (n=5) p<0.05, vehicle cocaine (n=15) vs PD325901 2.5 mg/kg (n=15), p<0.0001, vehicle cocaine (n=15) vs PD325901 5 mg/kg (n=5), p<0.01, vehicle cocaine (n=15) vs PD325901 10 mg/kg (n=5), p<0.01, vehicle cocaine (n=15) vs PD325901 25 mg/kg (n=5), p<0.01. *p<0.05, **p<0.01, ****p<0.0001, all the statistical significances are referred to the vehicle cocaine group indicated by the arrow. Data are shown as mean with SEM. DOI: http://dx.doi.org/10.7554/eLife.17111.010

Article Snippet: 1 hr after blocking in 5% normal goat serum and 0.1% Triton X-100 solution, slices were incubated overnight at 4°C with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) (1:200, Cell Signaling Technology Cat# 4370L, RRID: AB_2297462 ).

Techniques: Saline, Injection, Immunohistochemistry

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) , Cell Signaling , Cat# 9101; RRID: AB_331646 , 1:4000.

Techniques: Western Blot, Immunostaining, Immunoprecipitation, Protease Inhibitor, Software, Imaging, Slice Preparation, Incubation