Journal: The Journal of Neuroscience

Article Title: A Farnesyltransferase Inhibitor Restores Cognitive Deficits inTsc2^+/–Mice through Inhibition of Rheb1

doi: 10.1523/jneurosci.0449-21.2022

Figure Lengend Snippet: Figure 1. Treatment with FTIs and heterozygous knockout of Rheb1 restored spine synapse formation in Tsc21/ neurons. a, Confocal images of EGFP-expressing cultured hippocampal neu- rons from WT or Tsc21/ mouse. Immunodetection of VGluT11 (red) presynaptic boutons on EGFP (green)-expressing WT and Tsc21/ neurons treated with DMSO (vehicle) or 2 mM lonafar- nib. Scale bar, 5 mm. b, Cumulative probability distributions of the spine widths (left) and lengths (right) in a. Note that lonafarnib treatment increased the dendritic spine width (D= 0.5180; ***p , 0.0001) and decreased the dendritic spine length (D= 0.247; ***p , 0.001) compared with vehicle treatment in Tsc21/ neurons [spines/neurons, respectively; n = 541/12 (WT/vehi- cle), n = 363/10 (WT/lonafarnib), n = 525/12 (Tsc21//vehicle), and n = 328/8 (Tsc21//lonafarnib)]. c, No significant differences were observed in spine density in the condition of a [den- drites; n = 12 (WT, vehicle), n = 10 (WT, lonafarnib), n = 12 (Tsc21/, vehicle), and n = 8 (Tsc21/, lonafarnib); one-way ANOVA: F(3,38) = 0.996, p = 0.405; two-way ANOVA: treatment genotype interaction: F(1,38) = 0.078, p = 0.781; genotype: F(1,38) = 2.948, p = 0.094; treatment: F(1,38) = 0.008, p = 0.931]. d, Quantification of VGluT11 spines in a. The decrease of spine syn- apses in Tsc21/ was recovered by lonafarnib treatment [dendrites; n = 11 (WT, vehicle), n = 10 (WT, lonafarnib), n = 12 (Tsc21/, vehicle), and n = 12 (Tsc21/, lonafarnib); one-way ANOVA, post hoc Tukey’s test, F(3,41) = 36.13; ***p , 0.001, Tsc21/ 1 vehicle vs WT 1 vehicle; ***p , 0.001, Tsc21/ 1 vehicle vs Tsc21/ 1 lonafarnib; two-way ANOVA: treatment genotype interaction: F(1,41) = 32.93, p , 0.001; genotype: F(1,41) = 38.15, p , 0.001; treatment: F(1,41) = 32.50, p , 0.001]. e, Confocal images of EGFP-expressing cultured hippocampal neurons from a WT or Tsc21/ mouse. Immunodetection of VGluT11 (red) presynaptic boutons on EGFP (green)-expressing WT and Tsc21/ neurons treated with DMSO (vehicle) or 100 nM rapamycin. Scale bar, 5 mm. f, Cumulative probability plots of the dendritic spine widths (left) and lengths (right) in e. Note that rapamycin treatment increased the dendritic spine length (D= 0.086, *p = 0.030) and did not alter the dendritic spine width (D= 0.028, p = 0.982) compared with those following DMSO treatment in Tsc21/ neurons [spines/neurons, respectively; n = 570/20 (WT/vehicle), n = 564/17 (WT/rapamycin), n = 579/18 (Tsc21//vehicle), and n = 551/18 (Tsc21//rapamycin); Kolmogorov–Smirnov test]. g, No significant differences were observed in spine density in the condition of e [dendrites; n = 20 (WT, vehicle), n = 17 (WT, rapamycin), n = 18 (Tsc21/, vehicle), and n = 18 (Tsc21/, rapamycin); one-way ANOVA:

Article Snippet: The resultant soluble and insoluble fractions were immunoblotted as described previously (Sugiura et al., 2015), and primary antibodies against FLAG (1:800; catalog #F3165, Sigma-Aldrich), Rheb1 [1:1000; catalog #MAB3426, R&D Systems (recognizing whole Rheb); 1:1000; catalog #13879; Cell Signaling Technology (preferably recognizing unfarnesylated Rheb)], a/b -tubulin (1:1000; catalog #2148, Cell Signaling Technology), phospho (p)-ERK1/2 (Thr202/Tyr204; 1:1000; catalog #9101, Cell Signaling Technology), ERK1/2 (1:1000; catalog #9102, Cell Signaling Technology), syntenin1 (1:400; catalog #sc-100336, Santa Cruz Biotechnology), p-S6 (Ser240/244; 1:1000; catalog #5364, Cell Signaling Technology), and S6 (1:1000; catalog #2317, Cell Signaling Technology), and HRP-conjugated secondary antibodies (1:6000; catalog #170–6515, BIO-RAD) were used.

Techniques: Knock-Out, Expressing, Cell Culture, Immunodetection

Journal: The Journal of Neuroscience

Article Title: A Farnesyltransferase Inhibitor Restores Cognitive Deficits inTsc2^+/–Mice through Inhibition of Rheb1

doi: 10.1523/jneurosci.0449-21.2022

Figure Lengend Snippet: Figure 2. Rheb1 farnesylation and protein levels were modified by lonafarnib treatment. a, SDS-PAGE analyses of Rheb1 farnesylation. HEK293 T cells transfected with FLAG-Rheb1 were incubated with DMSO (lanes 1 and 3) or 2 mM lonafarnib (lanes 2 and 4), and their lysates were separated into supernatant (sup) and pellet (ppt) fractions. Top, Unfarnesylated (U) and farne- sylated (F) FLAG-Rheb1 proteins are indicated. Bottom, Quantification of unfarnesylated and farnesylated FLAG-Rheb1 proteins in each fraction shown in the top panel (n = 8 independent experiments; two-tailed t test with Welch’s correction: t(13.84) = 15.090, ***p , 0.001, vehicle-treated condition vs lonafarnib-treated condition in sup-farnesylated; t(9.182) = 9.030, ***p , 0.001, vehicle-treated condition vs lonafarnib-treated condition in sup-unfarnesylated; and t(7.084) = 6.374, ***p , 0.001 vehicle-treated condition vs lonafarnib-treated ppt-farnesy- lated; three-way ANOVA: solubility treatment farnesylation interaction: F(1,56) = 105.1, p , 0.001; solubility treatment interaction: F(1,56) = 4.895, p = 0.031; solubility farnesyla- tion interaction: F(1,56) = 2.711, p = 0.105; treatment farnesylation interaction: F(1,56) = 288.8, p , 0.001; solubility: F(1,56) = 207.6, p , 0.001; treatment: F(1,56) = 22.35, p , 0.001; farnesylation: F(1,56) = 29.14, p , 0.001. b, Top, Lonafarnib treatment also increased unfarnesylation of endogenous Rheb1 protein in HEK293 T cells. Bottom, Quantification of unfarnesylated and farnesylated Rheb1 proteins in each homogenate shown in the top panel (n = 7 independent experiments, two-tailed t test with Welch’s correction: t(6.829) = 14.394, ***p , 0.001, vehi- cle-treated condition vs lonafarnib-treated condition farnesylated; t(6.004) = 11.953, ***p , 0.001, vehicle-treated condition vs lonafarnib-treated condition unfarnesylated; two-way ANOVA: treatment farnesylation interaction: F(1,24) = 326.2, p , 0.001; treatment: F(1,24) = 2.204, p = 0.151; farnesylation: F(1,24) = 10.56, p = 0.003). c, Top, Lonafarnib treatment drastically increased unfarnesylated Rheb1 levels in primary cultured hippocampal neurons. Bottom, Quantification of Rheb1 levels in each homogenate shown in the top panel (n = 7 independent experi- ments, two-tailed t test with Welch’s correction: t(11.943) = 12.186, ***p , 0.001). d, Lonafarnib did not influence the activation of mTORC1. Primary cultured hippocampal neurons were treated with DMSO or lonafarnib. Top, p-S6 levels in each supernatant were compared. Bottom, Quantification of the p-S6 levels in each homogenate shown in the top panel (n = 10 independ- ent experiments; two-tailed t test with Welch’s correction, t(15.620) = 0.529 p = 0.604). e, Lonafarnib did not affect the activation of ERK1/2. Top, Primary cultured hippocampal neurons were treated with DMSO or lonafarnib. Phospho-ERK1/2 levels in each supernatant were compared. Bottom, Quantification of phospho-ERK1/2 levels in each homogenate shown in the top panel

Article Snippet: The resultant soluble and insoluble fractions were immunoblotted as described previously (Sugiura et al., 2015), and primary antibodies against FLAG (1:800; catalog #F3165, Sigma-Aldrich), Rheb1 [1:1000; catalog #MAB3426, R&D Systems (recognizing whole Rheb); 1:1000; catalog #13879; Cell Signaling Technology (preferably recognizing unfarnesylated Rheb)], a/b -tubulin (1:1000; catalog #2148, Cell Signaling Technology), phospho (p)-ERK1/2 (Thr202/Tyr204; 1:1000; catalog #9101, Cell Signaling Technology), ERK1/2 (1:1000; catalog #9102, Cell Signaling Technology), syntenin1 (1:400; catalog #sc-100336, Santa Cruz Biotechnology), p-S6 (Ser240/244; 1:1000; catalog #5364, Cell Signaling Technology), and S6 (1:1000; catalog #2317, Cell Signaling Technology), and HRP-conjugated secondary antibodies (1:6000; catalog #170–6515, BIO-RAD) were used.

Techniques: Modification, SDS Page, Transfection, Incubation, Two Tailed Test, Solubility, Cell Culture, Activation Assay

Journal: The Journal of Neuroscience

Article Title: A Farnesyltransferase Inhibitor Restores Cognitive Deficits inTsc2^+/–Mice through Inhibition of Rheb1

doi: 10.1523/jneurosci.0449-21.2022

Figure Lengend Snippet: Figure 3. Lonafarnib treatment inhibited the farnesylation of a hippocampal protein and restored dendritic spine morphology in the hippocampi of Tsc21/ mice in vivo. a, Lonafarnib treat- ment increased unfarnesylated Rheb1 protein in the liver in vivo. Livers were dissected from WT mice treated with DMSO or lonafarnib. Top, Lonafarnib treatment drastically increased unfarne- sylated Rheb1 levels. Bottom, Quantification of Rheb1 levels in each homogenate shown in the top panel (n = 7 independent experiments; two-tailed t test with Welch’s correction: t(11.659) = 5.576, ***p , 0.001). b, Lonafarnib treatment did not show a significant increase in unfarnesylated Rheb1 levels in the hippocampus in vivo. Top, Hippocampi were dissected from WT mice treated with DMSO or lonafarnib. Bottom, Quantification of Rheb1 levels in each homogenate shown in the top panel (n = 8 independent experiments, two-tailed t test with Welch’s correction: t(10.950) = 1.020, p = 0.330). c, Lonafarnib treatment increased unfarnesylated HDJ-2 protein in the hippocampus in vivo. Top, Hippocampi were dissected from WT mice treated with DMSO or lonafarnib. The HDJ-2 protein in each homogenate was immunoblotted. Unfarnesylated (U) and farnesylated (F) HDJ-2 proteins are indicated. Bottom, Quantification of the unfarnesylated HDJ- 2 protein levels shown in the top panel (n=7 independent experiments, two-tailed t test with Welch’s correction: t(10.860) = 3.795; ***p= 0.003). d, Lonafarnib treatment did not affect p-S6 pro- tein levels in the brain. Top, Phosphorylated and total S6 proteins are indicated. Bottom, Quantification of the phospho-S6 levels shown in the top panel (n=9 independent experiments; two-tailed t test with Welch’s correction: t(14.326) = 0.376, p=0.713). e, Lonafarnib treatment did not affect the phosphorylation of MAP kinase in the brain. Top, Phosphorylated and total ERK1/2 proteins are indicated. Bottom, Quantification of the phospho-ERK1/2 levels shown in the top panel (n=9 independent experiments; two-tailed t test with Welch’s correction: t(14.679) = 0.169, p=0.868). f, Representative images of S6 (green) and p-S6 (red) staining in the DG of vehicle- or lonafarnib-administered WT mice. Scale bar, 300mm. g, Quantification of relative p-S6/S6 intensity in the DG. Lonafarnib administration did not alter p-S6/S6 value in WT mice (n=18/3 hippocampi/animals; two-tailed t test with Welch’s correction: t(3.13153) = 1.899, p=0.150). Data are averaged and pre- sented by mice. h, Representative DiI-labeled dendrites from the hippocampal CA1 neurons of WT and Tsc21/ mice treated with lonafarnib or vehicle. Scale bar, 2mm. i, Cumulative probabil- ity plots of the spine width (left) and spine length (right) in h. Tsc21/ neurons showed a decreased spine width [D= 0.316; ***p , 0.001 (Tsc21/ 1 vehicle vs WT 1 vehicle)], and lonafarnib treatment rescued the aberrant spine shape [D= 0.2590, ***p , 0.001, Tsc21/ 1 vehicle vs Tsc21/ 1 lonafarnib; spines/neurons/animals, respectively: n = 1055/31/3 (WT 1 vehicle), n = 1155/30/3 (WT 1 lonafarnib), n = 1092/30/3 (Tsc21/ 1 vehicle), and n = 1039/32/3 (Tsc21/ 1 lonafarnib); Kolmogorov–Smirnov test]. j, Quantification of spine density in f [neurons/animals, respectively: n = 31/3 (WT, vehicle), 30/3 (WT, lonafarnib), n = 30/3 (Tsc21/, vehicle), and n = 32/3 (Tsc21/, lonafarnib); one-way ANOVA, F(3,8) = 0.195 p = 0.897; two-way ANOVA: treatment genotype interaction: F(1,8) = 0.361, p = 0.565; genotype: F(1,8) = 0.098, p = 0.763; treatment: F(1,8) = 0.126, p = 0.732]. Data are averaged and presented by mouse type. Error bars indicate SD. ***p , 0.005, n.s.: not significant. D: the largest absolute difference between the two distribution functions across all values.

Article Snippet: The resultant soluble and insoluble fractions were immunoblotted as described previously (Sugiura et al., 2015), and primary antibodies against FLAG (1:800; catalog #F3165, Sigma-Aldrich), Rheb1 [1:1000; catalog #MAB3426, R&D Systems (recognizing whole Rheb); 1:1000; catalog #13879; Cell Signaling Technology (preferably recognizing unfarnesylated Rheb)], a/b -tubulin (1:1000; catalog #2148, Cell Signaling Technology), phospho (p)-ERK1/2 (Thr202/Tyr204; 1:1000; catalog #9101, Cell Signaling Technology), ERK1/2 (1:1000; catalog #9102, Cell Signaling Technology), syntenin1 (1:400; catalog #sc-100336, Santa Cruz Biotechnology), p-S6 (Ser240/244; 1:1000; catalog #5364, Cell Signaling Technology), and S6 (1:1000; catalog #2317, Cell Signaling Technology), and HRP-conjugated secondary antibodies (1:6000; catalog #170–6515, BIO-RAD) were used.

Techniques: In Vivo, Two Tailed Test, Phospho-proteomics, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: A Farnesyltransferase Inhibitor Restores Cognitive Deficits inTsc2^+/–Mice through Inhibition of Rheb1

doi: 10.1523/jneurosci.0449-21.2022

Figure Lengend Snippet: Figure 7. Heterozygous knockout of Rheb1 mimicked the lonafarnib-mediated recovery of spine malformation in Tsc21/ neurons and impaired fear memory in Tsc21/ mice. a, Confocal images of cultured hippocampal neurons from CamkII-Cre, CamkII-Cre; Rheb1flox/1 (Rheb1-flox), Tsc21/; CamkII-Cre (Tsc21/; Cre), or Tsc21/; CamkII-Cre; Rheb1flox/1 (Tsc21/; Rheb1-flox) mice. Immunodetection of VGluT11 (red) presynaptic boutons on EGFP-expressing (green) neurons. Scale bar, 5 mm. b, Cumulative probability distributions of the spine widths (left) and lengths (right) in a. Note that heterozygous Rheb1 knockout increased the dendritic spine width (D= 0.281, ***p , 0.001) and decreased the dendritic spine length (D= 0.095, *p = 0.013) in Tsc21/ neurons [spines/neurons, respectively; n = 564/16 (CamkII-Cre), n = 558/14 (Rheb1-flox), n = 552/15 (Tsc21/; Cre), and n = 562/17 (Tsc21/; Rheb1-flox; Kolmogorov–Smirnov test)]. c, Quantification of spine density in a [dendrites; n = 16 (CamkII-Cre), n = 14 (Rheb1-flox), n = 15 (Tsc21/; Cre), and n = 17 (Tsc21/; Rheb1-flox); one-way ANOVA, post hoc Tukey’s test: F(3,58) = 0.548, p = 0.053 (CamkII-Cre vs Tsc21/; Cre)]. d, Quantification of VGluT11 spines in a. Decrease of spine synapses with VGluT1 puncta in Tsc21/; Cre neuron was restored in Tsc21/; Rheb1-flox neurons [dendrites; n = 8 (CamkII-Cre), n = 8 (Rheb1-flox), n = 10 (Tsc21/; Cre), and n = 10 (Tsc21/; Rheb1-flox); one-way ANOVA: post hoc Tukey’s test: F(3,32) = 14.061; ***p , 0.001, Tsc21/; Cre vs CamkII-Cre; ***p , 0.001, Tsc21/; Cre vs Rheb1-flox; ***p = 0.003, Tsc21/; Cre vs Tsc21/; Rheb1-flox]. e, Heterozygous Rheb1 knockout rescued the memory deficits of Tsc21/ mice. CamkII-Cre mice showed intact memory by freezing more in the familiar context than in the novel context (n = 12 animals; t(21.315) = 3.648, ***p = 0.002), whereas Tsc21/; Cre mice showed impaired contextual discrimination (n = 12 animals; t(15.688) = 1.908, p = 0.075). Heterozygous Rheb1 knockout rescued the deficit in con- textual discrimination (n = 12 animals; t(20.108) = 2.797, *p = 0.011), and Rheb1-flox mice exhibited normal memory (n = 12 animals; t(21.998) = 2.678, *p = 0.014), two-tailed t test with Welch’s correction. Two-way ANOVA: genotype apparatus interaction: F(3,88) = 0.789, p= 0.503; genotype: F(3,88) = 1.143, p=0.336; apparatus: F(1,88) = 30.88, p, 0.001). f, The hot plate test indicated no significant difference in the time for a nociceptive response among CamkII-Cre, Rheb1-flox, Tsc21/; Cre, and Tsc21/; Rheb1-flox mice (one-way ANOVA; n=15 animals; F(3,56) = 0.169, p=0.917). g, Representative DiI-labeled dendrites from the hippocampal CA1 neurons of CamkII-Cre, Rheb1-flox, Tsc21/; Cre, or Tsc21/; Rheb1-flox mice. Scale bar, 2mm. h, Cumulative probability plots of the spine width (left) and spine length (right) in g. Tsc21/; Cre neurons showed a decreased spine width (D=0.227; ***p, 0.001, Tsc21/; Cre vs CamkII-Cre), and heterozy- gous Rheb1 knockout rescued the aberrant spine width [D=0.179; ***p, 0.001, Tsc21/; Cre vs Tsc21/; spines/neurons/animals, respectively; Rheb1-flox; n= 1035/26/3 (CamkII-Cre), n=1027/25/3 (Rheb1-flox), n=1016/29/3 (Tsc21/; Cre), and n=1029/30/3 (Tsc21/; Rheb1-flox); Kolmogorov–Smirnov test)]. i, Quantification of spine density in g [neurons/animals, respec- tively; n=26/3 (CamkII-Cre), n=25/3 (Rheb1-flox), n=29/3 (Tsc21/; Cre), and n= 30/3 (Tsc21/; Rheb1-flox); one-way ANOVA, post hoc Tukey’s test: F(3, 8) = 0.175 p= 0.910]. Data are aver- aged and presented by mice. Error bars indicate SD. *p , 0.05, ***p , 0.005, n.s.: not significant. D: the largest absolute difference between the two distribution functions across all values.

Article Snippet: The resultant soluble and insoluble fractions were immunoblotted as described previously (Sugiura et al., 2015), and primary antibodies against FLAG (1:800; catalog #F3165, Sigma-Aldrich), Rheb1 [1:1000; catalog #MAB3426, R&D Systems (recognizing whole Rheb); 1:1000; catalog #13879; Cell Signaling Technology (preferably recognizing unfarnesylated Rheb)], a/b -tubulin (1:1000; catalog #2148, Cell Signaling Technology), phospho (p)-ERK1/2 (Thr202/Tyr204; 1:1000; catalog #9101, Cell Signaling Technology), ERK1/2 (1:1000; catalog #9102, Cell Signaling Technology), syntenin1 (1:400; catalog #sc-100336, Santa Cruz Biotechnology), p-S6 (Ser240/244; 1:1000; catalog #5364, Cell Signaling Technology), and S6 (1:1000; catalog #2317, Cell Signaling Technology), and HRP-conjugated secondary antibodies (1:6000; catalog #170–6515, BIO-RAD) were used.

Techniques: Knock-Out, Cell Culture, Immunodetection, Expressing, Two Tailed Test, Hot Plate Test, Labeling

Journal: The Journal of Neuroscience

Article Title: A Farnesyltransferase Inhibitor Restores Cognitive Deficits inTsc2^+/–Mice through Inhibition of Rheb1

doi: 10.1523/jneurosci.0449-21.2022

Figure Lengend Snippet: Figure 8. Lonafarnib administration had no or weak effect on heterozygous knockout of Rheb1 in the recovery of spine malfor- mation in Tsc21/ neurons and impaired fear memory in Tsc21/ mice. a, Representative DiI-labeled dendrites from the hippo- campal CA1 neurons of vehicle- or lonafarnib-administered Tsc21/; CamkII-Cre (Tsc21/; Cre) and Tsc21/; CamkII-Cre; Rheb1flox/1 (Tsc21/; Rheb1-flox) mice. Scale bar, 2mm. b, Cumulative probability plots of the spine width (left) and spine length (right) in a. Tsc21/; Rheb1-flox 1 lonafarnib neurons showed a comparable spine width to Tsc21/; Cre 1 lonafarnib neurons and Tsc21/; Rheb1-flox 1 vehicle neurons [D= 0.033, p = 0.638 (Tsc21/; Rheb1-flox 1 lonafarnib vs Tsc21/; Cre 1 lonafarnib); D = 0.009, ***p , 0.001 (Tsc21/; Rheb1-flox 1 lonafarnib vs Tsc21/; Rheb1-flox 1 vehicle; Tsc21/; Rheb1-flox 1 vehicle showed wider average; spines/neurons/animals, respectively: n = 1038/26/3 (Tsc21/; Cre 1 vehicle), n = 1027/26/3 (Tsc21/; Cre 1 lonafarnib), n = 1008/29/3 (Tsc21/; Rheb1-flox 1 vehicle), and n = 1023/26/3 (Tsc21/; Rheb1-flox 1 lonafarnib); Kolmogorov–Smirnov test]. c, Quantification of spine density in a [neurons/animals, respectively; n = 26/3 (Tsc21/; Cre 1 vehicle), n = 26/3 (Tsc21/; Cre 1 lonafarnib), n = 29/3 (Tsc21/; Rheb1-flox 1 vehicle), and n = 26/3 (Tsc21/; Rheb1-flox 1 lonafarnib); one-way ANOVA: F(3,8) = 1.440, p = 0.302; two-way ANOVA, treatment geno- type interaction: F(1,8) = 0.071, p = 0.797; genotype: F(1,8) = 3.418, p = 0.102; treatment: F(1,8) = 0.830, p = 0.389; data are averaged and presented by mouse type]. d, Schematic illustration of the experimental design for the contextual learning task. Tsc21/; Rheb1-flox 1 lonafarnib mice showed a comparable phenotype of learning ability to Tsc21/; Cre 1 lonafarnib mice and Tsc21/; Rheb1-flox 1 vehicle mice (n = 12 animals; two-tailed t test with Welch’s correction: t(21.225) = 0.483, p = 0.638 (Tsc21/; Cre 1 vehicle); t(21.359) = 2.149, *p = 0.043 (Tsc21/; Cre 1 lonafarnib); t(20.661) = 2.444, *p = 0.024 (Tsc21/; Rheb1-flox 1 vehicle); t(21.799) = 2.462, *p = 0.022 (Tsc21/; Rheb1-flox 1 lonafarnib); three-way ANOVA: genotype treat- ment apparatus interaction: F(1,88) = 1.088, p = 0.300; genotype treatment interaction: F(1,88) = 1.235, p = 0.269; geno- type apparatus interaction: F(1,88) = 0.770, p = 0.383; treatment apparatus interaction: F(1,88) = 0.644, p = 0.424; genotype: F(1,88) = 1.000, p = 0.320; treatment: F(1,88) = 0.829, p = 0.365; apparatus: F(1,88) = 13.80, p , 0.001; multiple

Article Snippet: The resultant soluble and insoluble fractions were immunoblotted as described previously (Sugiura et al., 2015), and primary antibodies against FLAG (1:800; catalog #F3165, Sigma-Aldrich), Rheb1 [1:1000; catalog #MAB3426, R&D Systems (recognizing whole Rheb); 1:1000; catalog #13879; Cell Signaling Technology (preferably recognizing unfarnesylated Rheb)], a/b -tubulin (1:1000; catalog #2148, Cell Signaling Technology), phospho (p)-ERK1/2 (Thr202/Tyr204; 1:1000; catalog #9101, Cell Signaling Technology), ERK1/2 (1:1000; catalog #9102, Cell Signaling Technology), syntenin1 (1:400; catalog #sc-100336, Santa Cruz Biotechnology), p-S6 (Ser240/244; 1:1000; catalog #5364, Cell Signaling Technology), and S6 (1:1000; catalog #2317, Cell Signaling Technology), and HRP-conjugated secondary antibodies (1:6000; catalog #170–6515, BIO-RAD) were used.

Techniques: Knock-Out, Labeling, Two Tailed Test