Journal: Cell reports

Article Title: Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

doi: 10.1016/j.celrep.2022.110441

Figure Lengend Snippet: Figure 2. Human PDAC tumors with reduced Ga13 expression and tumors developing in the KPCGfl/fland KPCGfl/+ mice demonstrate increased mTOR signaling (A) Analysis of samples in cBioPortal with low and high expression of GNA13 for E-cadherin (CDH1), catenin-b1 (CTNNB1), claudin-7 (CLDN7), p-PDK1 (p-S241), mTOR, RPS6, and EEF2 at the protein level using the reverse-phase protein array (RPPA) data (n = 43 and 43, respectively). t test, mean ± SEM,*p % 0.05, **p % 0.01.

Article Snippet: Tissue sections were incubated with antibodies for: E-cadherin (Cell Signaling #3195, RRID: AB_2291471, 1:500), Cytokeratin 19 (Abcam ab52625, RRID: AB_2281020, 1:500), Ki67 (Cell Signaling #12202, RRID: AB_2620142, 1:1000), cleaved caspase-3 (Cell Signaling #9664, RRID: AB_2070042, 1:800), mTOR (Cell Signaling #2983, RRID: AB_2105622, 1:1000), p-mTOR (Abcam ab109268, RRID: AB_10888105, 1:2000), Cell Reports 38, 110441, March 1, 2022 e3 RPS6 (Cell Signaling #2217, RRID: AB_331355, 1:1000), and p-RPS6 (Cell Signaling #4858, RRID: AB_916156, 1:1000) overnight at 4 C. Antibody binding was detected using HRP-conjugated anti-rabbit secondary antibody and visualized using ImmPACT DAB Peroxidase Substrate kit (VectorLabs, Burlingame, CA).

Techniques: Expressing, Protein Array

Journal: Cell reports

Article Title: Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

doi: 10.1016/j.celrep.2022.110441

Figure Lengend Snippet: Figure 4. Ga13 loss sensitizes KPC tumors to the mTOR inhibitor rapamycin and reduces tumor growth in vivo (A) Syngeneic KPCG+/+ and KPCGfl/fltumors were analyzed for p-Pdk1, Pdk1, p-mTOR, mTOR, p-Rps6, and Rps6 by western blotting using Hsp90 as a loading control. (B and C) The KPCG+/+ and KPCGfl/flcells were implanted subcutaneously in the flank of B6 mice (6–8 weeks old) and treated with vehicle control or rapamycin (60 mg/kg daily; arrow indicates the start of treatment), once the tumors reached 100 mm3. The tumor sizes were measured using a caliper, harvested day 27,

Article Snippet: Tissue sections were incubated with antibodies for: E-cadherin (Cell Signaling #3195, RRID: AB_2291471, 1:500), Cytokeratin 19 (Abcam ab52625, RRID: AB_2281020, 1:500), Ki67 (Cell Signaling #12202, RRID: AB_2620142, 1:1000), cleaved caspase-3 (Cell Signaling #9664, RRID: AB_2070042, 1:800), mTOR (Cell Signaling #2983, RRID: AB_2105622, 1:1000), p-mTOR (Abcam ab109268, RRID: AB_10888105, 1:2000), Cell Reports 38, 110441, March 1, 2022 e3 RPS6 (Cell Signaling #2217, RRID: AB_331355, 1:1000), and p-RPS6 (Cell Signaling #4858, RRID: AB_916156, 1:1000) overnight at 4 C. Antibody binding was detected using HRP-conjugated anti-rabbit secondary antibody and visualized using ImmPACT DAB Peroxidase Substrate kit (VectorLabs, Burlingame, CA).

Techniques: In Vivo, Western Blot, Control

Journal: Molecular and cellular endocrinology

Article Title: Stimulatory Effect of Insulin on Theca-Interstitial Cell Proliferation and Cell Cycle Regulatory Proteins through MTORC1 Dependent Pathway

doi: 10.1016/j.mce.2012.12.004

Figure Lengend Snippet: Cells were treated with insulin (1μg/ml) for different time periods. Cells were lysed using RIPA buffer and subjected to Western blot analysis using phospho-specific antibodies for RPS6KB1(Thr389), RPS6(Ser235/236) and EIF4EBP1(Thr37/46). Protein loading was monitored by stripping and reprobing the same blot with RPS6KB1, RPS6 and EIF4EBP1 antibodies (A). The graphs (B, C and D) represent densitrometric scans of phospho RPS6KB1(Thr389), phospho RPS6 (Ser235/236) and phospho EIF4EBP1(Thr37/46) protein expression normalized for total RPS6KB1, RPS6 and EIF4EBP1, respectively. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent mean ± SE. *, P < 0.05; **, P < 0.01; and ***, P< 0.001 vs. 0 min insulin treatment. a, b and c represent significant differences compared with 5min insulin treatment (a, P < 0.05; b, P < 0.01 and c, P < 0.001, respectively). # and ## represent significant differences compared with 15min insulin treatment (#, P <0.05 and ##, P < 0.01, respectively). @ and @@ represent significant differences compared with 30 min insulin treatment (@, P < 0.05 and @@, P < 0.01, respectively).

Article Snippet: MTORC1 inhibitor, rapamycin and antibodies against phosphorylated RPS6KB1 (Thr 389 ), phospho- RPS6KB1 (Thr 421 /Ser 424 ), phospho-EIF4EBP1 (Thr 37 / 46 ), phosphorylated ribosomal protein S6 (Ser 235 / 236 ), total RPS6KB1, total RPS6, total EIF4EBP1, CCND3, CDK4 and the Mtor siRNA kit were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Stripping Membranes, Expressing

Journal: Molecular and cellular endocrinology

Article Title: Stimulatory Effect of Insulin on Theca-Interstitial Cell Proliferation and Cell Cycle Regulatory Proteins through MTORC1 Dependent Pathway

doi: 10.1016/j.mce.2012.12.004

Figure Lengend Snippet: T-I cells were pretreated without or with rapamycin (20 nM) for 1 h prior to treatment for 30 min with insulin (1μg/ml). Control groups were treated with vehicle (DMSO). Cells were lysed and subjected to Western blot analysis using phosphorylation site-specific antibodies for RPS6KB1(Thr389), RPS6KB1(Thr421/Ser424), RPS6 (Ser235/236) and EIF4EBP1(Thr37/46). The levels of RPS6KB1 or RPS6 protein were used as internal controls. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent mean ± SE. ***, P< 0.001 vs. control. c represent significant differences compared with insulin (c, P < 0.001). INS= insulin; R= rapamycin

Article Snippet: MTORC1 inhibitor, rapamycin and antibodies against phosphorylated RPS6KB1 (Thr 389 ), phospho- RPS6KB1 (Thr 421 /Ser 424 ), phospho-EIF4EBP1 (Thr 37 / 46 ), phosphorylated ribosomal protein S6 (Ser 235 / 236 ), total RPS6KB1, total RPS6, total EIF4EBP1, CCND3, CDK4 and the Mtor siRNA kit were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot

Journal: Molecular and cellular endocrinology

Article Title: Stimulatory Effect of Insulin on Theca-Interstitial Cell Proliferation and Cell Cycle Regulatory Proteins through MTORC1 Dependent Pathway

doi: 10.1016/j.mce.2012.12.004

Figure Lengend Snippet: T-I cells were transfected with 100 nM of control siRNA or Mtor siRNA for 48 h and then treated without or with insulin (1μg/ml) for 30 min. Proteins (30 μg) were separated by SDS-PAGE (4–20%), and then immunoblotted with MTOR, phospho-specific RPS6KB1 (Thr389), RPS6KB1 (Thr421/Ser424), RPS6 (Ser235/236) and EIF4EBP1(Thr37/46) antibodies. Protein loading was monitored by stripping and reprobing the same blot with antibody for GAPDH. The graphs (B, C, D and E) represent densitrometric scans of MTOR, phospho RPS6KB1(Thr389), phospho RPS6 (Ser235/236) and phospho EIF4EBP1(Thr37/46) protein expression normalized for GAPDH, respectively. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent mean ± SE. *, P < 0.05; **, P < 0.01; and ***, P< 0.001 vs. control siRNA. a, b and c represent significant differences compared with control siRNA plus insulin treatment (a, P < 0.05; b, P < 0.01 and c, P < 0.001, respectively). INS= insulin

Article Snippet: MTORC1 inhibitor, rapamycin and antibodies against phosphorylated RPS6KB1 (Thr 389 ), phospho- RPS6KB1 (Thr 421 /Ser 424 ), phospho-EIF4EBP1 (Thr 37 / 46 ), phosphorylated ribosomal protein S6 (Ser 235 / 236 ), total RPS6KB1, total RPS6, total EIF4EBP1, CCND3, CDK4 and the Mtor siRNA kit were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Transfection, SDS Page, Stripping Membranes, Expressing

Journal: Molecular and cellular endocrinology

Article Title: Stimulatory Effect of Insulin on Theca-Interstitial Cell Proliferation and Cell Cycle Regulatory Proteins through MTORC1 Dependent Pathway

doi: 10.1016/j.mce.2012.12.004

Figure Lengend Snippet: Cells were pretreated without or with the PI3-kinase inhibitor, Wortmannin (100 nM) for 30 min or the MEK inhibitor, U0126 (10μM) for 1 h, followed by insulin (1μg/ml) treatment for 30 min. Control groups were treated with vehicle (DMSO). The cell lysates were examined for phospho-specific RPS6KB1(Thr389) and RPS6 (Ser235/236) by Western blot. Protein loading was monitored by stripping and reprobing the same blot with RPS6 antibody. The blot is representative of one experiment, and the graph represents the mean of three experiments. Error bars represent mean ± SE. *, P < 0.05; P **, P < 0.01; and ***, P< 0.001 vs. control. b and c represent significant differences compared with insulin (b, P < 0.01 and c, P < 0.001 respectively). # and ## represent significant differences compared with INS + W (#, P < 0.05 and ##, P < 0.01, respectively). INS= insulin; W= Wortmannin

Article Snippet: MTORC1 inhibitor, rapamycin and antibodies against phosphorylated RPS6KB1 (Thr 389 ), phospho- RPS6KB1 (Thr 421 /Ser 424 ), phospho-EIF4EBP1 (Thr 37 / 46 ), phosphorylated ribosomal protein S6 (Ser 235 / 236 ), total RPS6KB1, total RPS6, total EIF4EBP1, CCND3, CDK4 and the Mtor siRNA kit were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Stripping Membranes

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Marmoset as a Model to Study Kidney Changes Associated With Aging

doi: 10.1093/gerona/gly237

Figure Lengend Snippet: Signaling pathways are dysregulated in the kidney cortex of aged male but not female marmosets. Renal cortical lysates were employed to perform immunoblotting using antibodies against (A) phospho-ACC, ACC, (B) phospho-rpS6, rpS6, (C) Tyr-1165/1166 phosphorylated IGF-1 receptor (P-IGF 1R), IGF 1R, (E) Ser-9 phosphorylated GSK 3β, GSK 3β, (F) Klotho and actin. (D) ELISA was performed to assess changes in Tyr-1150/1151 phosphorylation of the IR (Y = young, A = aged). Data are shown as mean ± SEM, n = 4 young male and n = 4 young female marmosets, n = 4–5 aged male and n = 4–5 aged female marmosets (*p < .05, **p < .01, ***p < .001 vs young male; #p < .05 vs young female, $$$p < .001 vs aged male by two-way ANOVA).

Article Snippet: We employed antibodies against the following: Laminin γ1, phospho-Tyr1165/1166-IGF-1 receptor, IGF-1 receptor, phospho-Ser9-glycogen synthase kinase 3 β (GSK3β), GSK3α/β (binds both isoforms), TGFβ1, CSE, CBS (Santa Cruz Biotechnology, Inc., Dallas, TX), type III collagen, fibronectin, nephrin (Abcam Plc, Cambridge, MA), phospho-Ser79-Acetyl-CoA carboxylase (ACC), ACC, phospho-Ser235/236-ribosomal protein S6 (rpS6), rpS6, phospho-Ser423/425-Smad3 (Cell Signaling Technology, Inc., Denvers, MA), Smad3 (Thermo Fisher Scientific, Inc., Waltham, MA), actin (Millipore Sigma, St. Louis, MO).

Techniques: Protein-Protein interactions, Western Blot, Enzyme-linked Immunosorbent Assay, Phospho-proteomics