Structured Review

Cell Signaling Technology Inc phospho akt s473
Arsenic growth inhibition correlates with decreased temsirolimus-induced <t>phospho-AKT.</t> MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for <t>phospho-S473-AKT,</t> phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.
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1) Product Images from "Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer"

Article Title: Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085995

Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT. MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.
Figure Legend Snippet: Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT. MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.

Techniques Used: Inhibition, Multiple Displacement Amplification, Western Blot

Addition of ATO decreases rapamycin-induced AKT activation in vivo . Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).
Figure Legend Snippet: Addition of ATO decreases rapamycin-induced AKT activation in vivo . Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).

Techniques Used: Activation Assay, In Vivo, Staining, Software, Mouse Assay

2) Product Images from "Depletion of Rictor, an essential protein component of mTORC2, decreases male lifespan"

Article Title: Depletion of Rictor, an essential protein component of mTORC2, decreases male lifespan

Journal: Aging Cell

doi: 10.1111/acel.12256

Analysis of mTOR signaling in the livers of L-RKO and rictor +/− mice. (A,B) Analysis of protein expression and phosphorylation in the livers of wild-type and (A) rictor +/− and (B) L-RKO mice fasted overnight and then refed for 3 h. (C) Quantification of the expression of RICTOR and AKT relative to β-tubulin, and the phosphorylation of AKT S473, T450, and T308 relative to AKT. Analysis was of 10-week-old mice ( n = 14 wild-type, 8 rictor +/− and 7 L-RKO males; 12 wild-type, 6 rictor +/− and 6 L-RKO females, * = P
Figure Legend Snippet: Analysis of mTOR signaling in the livers of L-RKO and rictor +/− mice. (A,B) Analysis of protein expression and phosphorylation in the livers of wild-type and (A) rictor +/− and (B) L-RKO mice fasted overnight and then refed for 3 h. (C) Quantification of the expression of RICTOR and AKT relative to β-tubulin, and the phosphorylation of AKT S473, T450, and T308 relative to AKT. Analysis was of 10-week-old mice ( n = 14 wild-type, 8 rictor +/− and 7 L-RKO males; 12 wild-type, 6 rictor +/− and 6 L-RKO females, * = P

Techniques Used: Mouse Assay, Expressing

3) Product Images from "Restriction of dietary protein decreases mTORC1 in tumors and somatic tissues of a tumor-bearing mouse xenograft model"

Article Title: Restriction of dietary protein decreases mTORC1 in tumors and somatic tissues of a tumor-bearing mouse xenograft model

Journal: Oncotarget

doi:

A protein restricted diet inhibits mTORC1 signaling in tumors A. Tissue lysates from tumor xenografts were examined for phosphorylation of S6 S240/S244 and AKT S473 by western blotting. B. Quantification of S6 and AKT phosphorylation, normalized to total S6 or AKT protein, was performed using NIH ImageJ. C. Quantification of S6, normalized to β-TUBULIN, was performed using NIH Image J ( n = 7–10 tumors per group, * = p
Figure Legend Snippet: A protein restricted diet inhibits mTORC1 signaling in tumors A. Tissue lysates from tumor xenografts were examined for phosphorylation of S6 S240/S244 and AKT S473 by western blotting. B. Quantification of S6 and AKT phosphorylation, normalized to total S6 or AKT protein, was performed using NIH ImageJ. C. Quantification of S6, normalized to β-TUBULIN, was performed using NIH Image J ( n = 7–10 tumors per group, * = p

Techniques Used: Western Blot

A protein restricted diet inhibits mTORC1 signaling in vivo Tissue lysates from A. liver, B. muscle, C. heart and D. adipose tissue were examined for phosphorylation of S6 S240/S244 and AKT S473 by western blotting. Quantification of S6 and AKT phosphorylation, normalized to total S6 or AKT protein, was performed using NIH ImageJ ( n = 6–7 samples per group, means with the same letter are not significantly different from each other (Tukey–Kramer test following ANOVA, p
Figure Legend Snippet: A protein restricted diet inhibits mTORC1 signaling in vivo Tissue lysates from A. liver, B. muscle, C. heart and D. adipose tissue were examined for phosphorylation of S6 S240/S244 and AKT S473 by western blotting. Quantification of S6 and AKT phosphorylation, normalized to total S6 or AKT protein, was performed using NIH ImageJ ( n = 6–7 samples per group, means with the same letter are not significantly different from each other (Tukey–Kramer test following ANOVA, p

Techniques Used: In Vivo, Western Blot

4) Product Images from "mTORC1 Signaling in Oocytes Is Dispensable for the Survival of Primordial Follicles and for Female Fertility"

Article Title: mTORC1 Signaling in Oocytes Is Dispensable for the Survival of Primordial Follicles and for Female Fertility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0110491

PI3K–Akt signaling in Oo Rptor −/− and Oo Rptor +/+ oocytes. Oocytes were isolated from the ovaries of Oo Rptor −/− and Oo Rptor +/+ mice at PD12–14, and western blots were performed as described in the Materials and Methods . Levels of phosphorylation of Akt at S473 and T308 are elevated in Oo Rptor −/− oocytes compared to Oo Rptor +/+ oocytes, and this indicates that PI3K–Akt signaling in Oo Rptor −/− oocytes is enhanced. Levels of total Akt and β-actin were used as internal controls.
Figure Legend Snippet: PI3K–Akt signaling in Oo Rptor −/− and Oo Rptor +/+ oocytes. Oocytes were isolated from the ovaries of Oo Rptor −/− and Oo Rptor +/+ mice at PD12–14, and western blots were performed as described in the Materials and Methods . Levels of phosphorylation of Akt at S473 and T308 are elevated in Oo Rptor −/− oocytes compared to Oo Rptor +/+ oocytes, and this indicates that PI3K–Akt signaling in Oo Rptor −/− oocytes is enhanced. Levels of total Akt and β-actin were used as internal controls.

Techniques Used: Isolation, Mouse Assay, Western Blot

5) Product Images from "Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer"

Article Title: Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085995

Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT. MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.
Figure Legend Snippet: Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT. MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.

Techniques Used: Inhibition, Multiple Displacement Amplification, Western Blot

Addition of ATO decreases rapamycin-induced AKT activation in vivo . Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).
Figure Legend Snippet: Addition of ATO decreases rapamycin-induced AKT activation in vivo . Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).

Techniques Used: Activation Assay, In Vivo, Staining, Software, Mouse Assay

6) Product Images from "Insulin regulates liver metabolism in vivo in the absence of hepatic Akt and Foxo1"

Article Title: Insulin regulates liver metabolism in vivo in the absence of hepatic Akt and Foxo1

Journal: Nature medicine

doi: 10.1038/nm.2686

Aberrant insulin signaling and disrupted expression of Foxo1–regulated genes in the DLKO livers but not in the 2LKO livers ( a ) Western blot of Akt1 and Akt2 in primary hepatocytes, muscle and adipose tissues from the DLWT and DLKO mice. ( b ) Western blot of liver lysates. p–Akt 473: phosphorylation of Akt at S473. p–S6: phosphorylation of ribosomal protein S6 at S240, S244. p–Gsk3: phosphorylation of Gsk3α at S21 and Gsk3β at S9. p–Erk: phosphorylation of Erk at T202 and Y204. ( c ) Relative expression of genes in livers quantitated by real time PCR. In ( b ) and ( c ), mice were either fasted overnight (open bars) or fasted overnight followed by 4 hours feeding with normal chow (filled bars).
Figure Legend Snippet: Aberrant insulin signaling and disrupted expression of Foxo1–regulated genes in the DLKO livers but not in the 2LKO livers ( a ) Western blot of Akt1 and Akt2 in primary hepatocytes, muscle and adipose tissues from the DLWT and DLKO mice. ( b ) Western blot of liver lysates. p–Akt 473: phosphorylation of Akt at S473. p–S6: phosphorylation of ribosomal protein S6 at S240, S244. p–Gsk3: phosphorylation of Gsk3α at S21 and Gsk3β at S9. p–Erk: phosphorylation of Erk at T202 and Y204. ( c ) Relative expression of genes in livers quantitated by real time PCR. In ( b ) and ( c ), mice were either fasted overnight (open bars) or fasted overnight followed by 4 hours feeding with normal chow (filled bars).

Techniques Used: Expressing, Western Blot, Mouse Assay, Real-time Polymerase Chain Reaction

7) Product Images from "ARG2 impairs endothelial autophagy through regulation of MTOR and PRKAA/AMPK signaling in advanced atherosclerosis"

Article Title: ARG2 impairs endothelial autophagy through regulation of MTOR and PRKAA/AMPK signaling in advanced atherosclerosis

Journal: Autophagy

doi: 10.4161/15548627.2014.981789

ARG2 enhances TP53, and MTORC2-AKT-MTORC1-RPS6KB1 and impairs PRKAA signaling independently of its enzymatic activity. Young HUVECs were transduced as in Fig.1A. Sixty-4 h post transduction, cell lysates were prepared and subjected to ( A ) immunoblotting analysis of RICTOR, PRKAA-T172, and PRKAA, AKT-S473, and AKT, TP53-S15 and TP53, RPS6-S235/236 and RPS6, and tubulin. (( B ) to F ) Quantification of the signals is also shown (n = 4 as indicated in the figure). * P
Figure Legend Snippet: ARG2 enhances TP53, and MTORC2-AKT-MTORC1-RPS6KB1 and impairs PRKAA signaling independently of its enzymatic activity. Young HUVECs were transduced as in Fig.1A. Sixty-4 h post transduction, cell lysates were prepared and subjected to ( A ) immunoblotting analysis of RICTOR, PRKAA-T172, and PRKAA, AKT-S473, and AKT, TP53-S15 and TP53, RPS6-S235/236 and RPS6, and tubulin. (( B ) to F ) Quantification of the signals is also shown (n = 4 as indicated in the figure). * P

Techniques Used: Activity Assay, Transduction

Silencing ARG2 in senescent HUVECs augments autophagy and PRKAA signaling while inhibiting RICTOR expression and activation of AKT and RPS6KB1. The senescent HUVECs were transduced either with rAd/U6- LacZ shRNA as control or rAd/U6- ARG2 shRNA . Eighty-8 h (serum-free during the last 16 h) post transduction, cells were subjected to ( A ) immunostaining of LC3-I/-II (red) followed by DAPI staining (blue). Shown are representative merged images from 6 independent experiments. Scale bar = 0.1 mm. (( B )and C ) Immunoblotting analysis LC3-I/-II, ATG12–ATG5, SQSTM1, PRKAA-T172, and total PRKAA, RPS6-S235/236 and total RPS6. RICTOR and tubulin, AKT-S473 and total AKT. Quantification of the immunoblotting signals is shown in the bar graphs in the right panels (n = 3 to 6 as indicated in the figures). * P
Figure Legend Snippet: Silencing ARG2 in senescent HUVECs augments autophagy and PRKAA signaling while inhibiting RICTOR expression and activation of AKT and RPS6KB1. The senescent HUVECs were transduced either with rAd/U6- LacZ shRNA as control or rAd/U6- ARG2 shRNA . Eighty-8 h (serum-free during the last 16 h) post transduction, cells were subjected to ( A ) immunostaining of LC3-I/-II (red) followed by DAPI staining (blue). Shown are representative merged images from 6 independent experiments. Scale bar = 0.1 mm. (( B )and C ) Immunoblotting analysis LC3-I/-II, ATG12–ATG5, SQSTM1, PRKAA-T172, and total PRKAA, RPS6-S235/236 and total RPS6. RICTOR and tubulin, AKT-S473 and total AKT. Quantification of the immunoblotting signals is shown in the bar graphs in the right panels (n = 3 to 6 as indicated in the figures). * P

Techniques Used: Expressing, Activation Assay, shRNA, Transduction, Immunostaining, Staining

Silencing RICTOR in senescent HUVECs enhances autophagy and PRKAA signaling while reducing ARG2 protein level and signaling of TP53, AKT, and RPS6KB1. The senescent HUVECs were transduced either with rAd/U6- LacZ shRNA as control or rAd/U6- RICTOR shRNA . Eighty-8 h (serum-free during the last 16 h) post transduction, cells were subjected to immunoblotting analysis of RICTOR, LC3-I/-II, ATG12–ATG5, SQSTM1, and tubulin, AKT-S473 and total AKT, RPS6-S235/236 and total RPS6, PRKAA-T172 and total PRKAA, TP53-S15 and total TP53, ARG2 and tubulin. Quantification of the immunoblotting signals is shown in the bar graphs in the right panels (n = 3 to 4 as indicated in the figures). * P
Figure Legend Snippet: Silencing RICTOR in senescent HUVECs enhances autophagy and PRKAA signaling while reducing ARG2 protein level and signaling of TP53, AKT, and RPS6KB1. The senescent HUVECs were transduced either with rAd/U6- LacZ shRNA as control or rAd/U6- RICTOR shRNA . Eighty-8 h (serum-free during the last 16 h) post transduction, cells were subjected to immunoblotting analysis of RICTOR, LC3-I/-II, ATG12–ATG5, SQSTM1, and tubulin, AKT-S473 and total AKT, RPS6-S235/236 and total RPS6, PRKAA-T172 and total PRKAA, TP53-S15 and total TP53, ARG2 and tubulin. Quantification of the immunoblotting signals is shown in the bar graphs in the right panels (n = 3 to 4 as indicated in the figures). * P

Techniques Used: shRNA, Transduction

8) Product Images from "Mcl-1 phosphorylation without degradation mediates sensitivity to HDAC inhibitors by liberating BH3-only proteins"

Article Title: Mcl-1 phosphorylation without degradation mediates sensitivity to HDAC inhibitors by liberating BH3-only proteins

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-18-0399

HDAC inhibitors promote GSK3β-mediated Mcl-1 phosphorylation and apoptosis. (A) Western blotting of indicated proteins in HCT116 cells treated with SAHA or MS-275 at indicated time points. p-Mcl-1: S159/T163; p-GSK3β: S9; p-ERK: T202/Y204; p-AKT: S473. (B) HCT116 cells were treated with SAHA with or without the GSK3 inhibitor SB216763 (5 µM) for 24 hr. Left , western blotting of indicated proteins; right , apoptosis was analyzed by counting condensed and fragmented nuclei after nuclear staining with Hoechst 33258. (C) Western blotting of Mcl-1 in HCT116 cells treated with indicated agents for 24 hr. ABT-263: 5 µM; ABT-737: 5 µM; SAHA: 4 µM; MS-275: 5 µM; regorafenib: 40 µM; sorafenib: 20 µM; UCN-01: 1 µM; sunitinib: 15 µM. (D) Western blotting of indicated proteins in HCT116 cells transfected with V5-tagged WT or 4A mutant Mcl-1 (S121A/E125A/S159A/T163A) and treated with SAHA or MS-275 for 24 hr. (E) Apoptosis in HCT116 cells transfected and treated as in (D) was analyzed as in (B). (F) Western blotting of indicated proteins in WT and Mcl-1 knock-in ( Mcl-1- KI) HCT116 cells treated with SAHA or MS-275 for 24 hr. In (A)-(F), SAHA: 4 µM; MS-275: 5 µM. In (B) and (E), results were expressed as means ± s.d. of three independent experiments. * , P
Figure Legend Snippet: HDAC inhibitors promote GSK3β-mediated Mcl-1 phosphorylation and apoptosis. (A) Western blotting of indicated proteins in HCT116 cells treated with SAHA or MS-275 at indicated time points. p-Mcl-1: S159/T163; p-GSK3β: S9; p-ERK: T202/Y204; p-AKT: S473. (B) HCT116 cells were treated with SAHA with or without the GSK3 inhibitor SB216763 (5 µM) for 24 hr. Left , western blotting of indicated proteins; right , apoptosis was analyzed by counting condensed and fragmented nuclei after nuclear staining with Hoechst 33258. (C) Western blotting of Mcl-1 in HCT116 cells treated with indicated agents for 24 hr. ABT-263: 5 µM; ABT-737: 5 µM; SAHA: 4 µM; MS-275: 5 µM; regorafenib: 40 µM; sorafenib: 20 µM; UCN-01: 1 µM; sunitinib: 15 µM. (D) Western blotting of indicated proteins in HCT116 cells transfected with V5-tagged WT or 4A mutant Mcl-1 (S121A/E125A/S159A/T163A) and treated with SAHA or MS-275 for 24 hr. (E) Apoptosis in HCT116 cells transfected and treated as in (D) was analyzed as in (B). (F) Western blotting of indicated proteins in WT and Mcl-1 knock-in ( Mcl-1- KI) HCT116 cells treated with SAHA or MS-275 for 24 hr. In (A)-(F), SAHA: 4 µM; MS-275: 5 µM. In (B) and (E), results were expressed as means ± s.d. of three independent experiments. * , P

Techniques Used: Western Blot, Mass Spectrometry, Staining, Transfection, Mutagenesis, Knock-In

9) Product Images from "Abrogation of PIK3CA or PIK3R1 reduces proliferation, migration, and invasion in glioblastoma multiforme cells"

Article Title: Abrogation of PIK3CA or PIK3R1 reduces proliferation, migration, and invasion in glioblastoma multiforme cells

Journal: Oncotarget

doi:

Confirmation of PIK3CA and PIK3R1 knockdown in cell lines A, PIK3CA and PIK3R1 class IA PI3K subunit expression was reduced using lentiviral-mediated shRNA targeted to each subunit specifically, and to each in combination. B, FAK activation at Y397 was observed for each cell line. C, Akt activation at T308 and S473 was observed, as was PTEN status.
Figure Legend Snippet: Confirmation of PIK3CA and PIK3R1 knockdown in cell lines A, PIK3CA and PIK3R1 class IA PI3K subunit expression was reduced using lentiviral-mediated shRNA targeted to each subunit specifically, and to each in combination. B, FAK activation at Y397 was observed for each cell line. C, Akt activation at T308 and S473 was observed, as was PTEN status.

Techniques Used: IA, Expressing, shRNA, Activation Assay

10) Product Images from "Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells"

Article Title: Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20131080

mTORC2 regulates COX-2 expression via PI3K–AKT pathway in TSC2-deficient cells. (a) Tsc2 −/− p53 −/− MEFs were treated with 20 nM rapamycin, 250 nM Torin 1, or 20 µM LY294002 for 24 h. Levels of COX-2, 4EBP1, and phospho-S6 (S235/236) were assessed by immunoblot. (b and c) Tsc2 −/− p53 −/− MEFs were transfected with Rictor shRNA or control shRNA, and then selected with puromycin to obtain stable cells. Control shRNA or with Rictor shRNA- Tsc2 −/− p53 −/− MEFs were treated with vehicle or 20 nM rapamycin for 24 h. Levels of COX-2, COX-1, phospho-S6 (S235/236), and phospho-Akt (S473) were assessed by immunoblot. (d) LAM patient–derived cells (TSC − ) cells were treated with 20 nM rapamycin, or 250 nM Torin 1, or 50 µM NS398 for 24 h. Levels of COX-2 and phospho-S6 (S235/236) were assessed by immunoblot. (e) TSC2-deficient LAM patient–derived cells were treated with 250 nM Torin 1 for 24 h. Levels of COX-2, phospho-p44/42–MAPK, phospho-Akt S473, and phospho-S6 (S235/236) were assessed by immunoblot. (f) TSC2-deficient LAM patient–derived cells were treated with 5 µM PI-103 or 5 µM AKTVIII for 24 h. Levels of COX-2 and phospho-Akt (S473) were assessed by immunoblot. (g) TSC2-deficient LAM patient–derived cells were treated with 50 µM PD98059, 5 µM PI-103, or PD98059 plus PI-103 for 24 h. Levels of COX-2, phospho-Akt S473, phospho-p44/42–MAPK, and phospho-S6 (S235/236) were assessed by immunoblot. (h) TSC2-deficient LAM patient–derived cells were treated with 1 µM Gefitinib or 1 µM Afatinib for 24 h. Levels of COX-2, EGFR, and phospho-Akt S473 were assessed by immunoblot. (i) ELT3 cells expressing empty vector (TSC − ) and TSC2-addback (TSC2 + ), or LAM patient–derived cells (TSC − ) cells and TSC2-addback cells (TSC2 + ) were treated with 20 nM rapamycin (Rapa) for 24 h. Subcellular localization of EGFR was examined using confocal microscopy. Bar, 10 µM. (j) Cells were treated with 50 µM Sulindac, 50 µM NS398, or 450 µM aspirin for 24 h. Levels of COX-1, COX-2, phospho-p44/42 MAPK, and phospho-S6 were assessed by immunoblot. (a–j) Results are representative of two to four different experiments. (k) PGE 2 levels from conditioned media were measured (ELISA). Results are representative of six sets of independent samples per group. (l) Cell proliferation after drug treatment was measured using MTT assay. Results are representative of 12 sets of independent samples per group. *, P
Figure Legend Snippet: mTORC2 regulates COX-2 expression via PI3K–AKT pathway in TSC2-deficient cells. (a) Tsc2 −/− p53 −/− MEFs were treated with 20 nM rapamycin, 250 nM Torin 1, or 20 µM LY294002 for 24 h. Levels of COX-2, 4EBP1, and phospho-S6 (S235/236) were assessed by immunoblot. (b and c) Tsc2 −/− p53 −/− MEFs were transfected with Rictor shRNA or control shRNA, and then selected with puromycin to obtain stable cells. Control shRNA or with Rictor shRNA- Tsc2 −/− p53 −/− MEFs were treated with vehicle or 20 nM rapamycin for 24 h. Levels of COX-2, COX-1, phospho-S6 (S235/236), and phospho-Akt (S473) were assessed by immunoblot. (d) LAM patient–derived cells (TSC − ) cells were treated with 20 nM rapamycin, or 250 nM Torin 1, or 50 µM NS398 for 24 h. Levels of COX-2 and phospho-S6 (S235/236) were assessed by immunoblot. (e) TSC2-deficient LAM patient–derived cells were treated with 250 nM Torin 1 for 24 h. Levels of COX-2, phospho-p44/42–MAPK, phospho-Akt S473, and phospho-S6 (S235/236) were assessed by immunoblot. (f) TSC2-deficient LAM patient–derived cells were treated with 5 µM PI-103 or 5 µM AKTVIII for 24 h. Levels of COX-2 and phospho-Akt (S473) were assessed by immunoblot. (g) TSC2-deficient LAM patient–derived cells were treated with 50 µM PD98059, 5 µM PI-103, or PD98059 plus PI-103 for 24 h. Levels of COX-2, phospho-Akt S473, phospho-p44/42–MAPK, and phospho-S6 (S235/236) were assessed by immunoblot. (h) TSC2-deficient LAM patient–derived cells were treated with 1 µM Gefitinib or 1 µM Afatinib for 24 h. Levels of COX-2, EGFR, and phospho-Akt S473 were assessed by immunoblot. (i) ELT3 cells expressing empty vector (TSC − ) and TSC2-addback (TSC2 + ), or LAM patient–derived cells (TSC − ) cells and TSC2-addback cells (TSC2 + ) were treated with 20 nM rapamycin (Rapa) for 24 h. Subcellular localization of EGFR was examined using confocal microscopy. Bar, 10 µM. (j) Cells were treated with 50 µM Sulindac, 50 µM NS398, or 450 µM aspirin for 24 h. Levels of COX-1, COX-2, phospho-p44/42 MAPK, and phospho-S6 were assessed by immunoblot. (a–j) Results are representative of two to four different experiments. (k) PGE 2 levels from conditioned media were measured (ELISA). Results are representative of six sets of independent samples per group. (l) Cell proliferation after drug treatment was measured using MTT assay. Results are representative of 12 sets of independent samples per group. *, P

Techniques Used: Expressing, Transfection, shRNA, Laser Capture Microdissection, Derivative Assay, Plasmid Preparation, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, MTT Assay

11) Product Images from "Sex‐ and tissue‐specific changes in mTOR signaling with age in C57 BL/6J mice"

Article Title: Sex‐ and tissue‐specific changes in mTOR signaling with age in C57 BL/6J mice

Journal: Aging Cell

doi: 10.1111/acel.12425

mTORC 2 activity in fasted male and female C57 BL /6J.Nia mice. (A–D) The phosphorylation of AKT was assessed by Western blotting of (A) liver, (B) muscle, (C) adipose, and (D) heart tissue lysates. Additional western blots included in the quantification are shown in Fig. S1B. Young refers to 6‐month‐old males and females (10 males, 5 females), Middle refers to 24‐month‐old males and 22‐month‐old females (10 males, 5 females), and Old refers to 30‐month‐old males and 26‐month‐old females (8 males, 4 females). Quantification of AKT T308 and S473 are relative to AKT (* P
Figure Legend Snippet: mTORC 2 activity in fasted male and female C57 BL /6J.Nia mice. (A–D) The phosphorylation of AKT was assessed by Western blotting of (A) liver, (B) muscle, (C) adipose, and (D) heart tissue lysates. Additional western blots included in the quantification are shown in Fig. S1B. Young refers to 6‐month‐old males and females (10 males, 5 females), Middle refers to 24‐month‐old males and 22‐month‐old females (10 males, 5 females), and Old refers to 30‐month‐old males and 26‐month‐old females (8 males, 4 females). Quantification of AKT T308 and S473 are relative to AKT (* P

Techniques Used: Activity Assay, Mouse Assay, Western Blot

mTORC 2 activity in refed male and female C57 BL /6J.Nia mice. (A–D) The phosphorylation of AKT was assessed by Western blotting of (A) liver, (B) muscle, (C) adipose, and (D) heart tissue lysates from mice that were fasted overnight and then refed for 45 min. Additional western blots included in the quantification are shown in Fig. S2B. Young refers to 6‐month‐old males and females (10 males, 5 females), Middle refers to 24‐month‐old males and 22‐month‐old females (10 males, 5 females), and Old refers to 30‐month‐old males and 26‐month‐old females (8 males, 4 females). Quantification of AKT T308 and S473 are relative to AKT (** P
Figure Legend Snippet: mTORC 2 activity in refed male and female C57 BL /6J.Nia mice. (A–D) The phosphorylation of AKT was assessed by Western blotting of (A) liver, (B) muscle, (C) adipose, and (D) heart tissue lysates from mice that were fasted overnight and then refed for 45 min. Additional western blots included in the quantification are shown in Fig. S2B. Young refers to 6‐month‐old males and females (10 males, 5 females), Middle refers to 24‐month‐old males and 22‐month‐old females (10 males, 5 females), and Old refers to 30‐month‐old males and 26‐month‐old females (8 males, 4 females). Quantification of AKT T308 and S473 are relative to AKT (** P

Techniques Used: Activity Assay, Mouse Assay, Western Blot

mTOR activity in male and female young C57 BL /6J.Nia mice. (A–D) The phosphorylation of S6, 4E‐ BP 1, and AKT was assessed by Western blotting of (A) liver, (B) muscle, (C) adipose, and (D) heart tissue from mice that were either fasted overnight (Fasted) or fasted overnight and then refed for 45 min (Refed). Additional western blots included in the quantification are shown in Fig. S3. Quantification of S6 S240/S244 is relative to S6, 4E‐ BP 1 T37/S46 is relative to 4E‐ BP 1 and AKT S473 is relative to AKT (** P
Figure Legend Snippet: mTOR activity in male and female young C57 BL /6J.Nia mice. (A–D) The phosphorylation of S6, 4E‐ BP 1, and AKT was assessed by Western blotting of (A) liver, (B) muscle, (C) adipose, and (D) heart tissue from mice that were either fasted overnight (Fasted) or fasted overnight and then refed for 45 min (Refed). Additional western blots included in the quantification are shown in Fig. S3. Quantification of S6 S240/S244 is relative to S6, 4E‐ BP 1 T37/S46 is relative to 4E‐ BP 1 and AKT S473 is relative to AKT (** P

Techniques Used: Activity Assay, Mouse Assay, Western Blot

12) Product Images from "Crosstalk of the EphA2 Receptor with a Serine/Threonine Phosphatase Suppresses the Akt-mTORC1 Pathway in Cancer Cells"

Article Title: Crosstalk of the EphA2 Receptor with a Serine/Threonine Phosphatase Suppresses the Akt-mTORC1 Pathway in Cancer Cells

Journal: Cellular signalling

doi: 10.1016/j.cellsig.2010.09.004

Serine/threonine phosphatase activity is required for EphA2-dependent Akt dephosphorylation. (A) Ephrin-A1 decreases Akt phosphorylation almost as much as the potent PI3 kinase inhibitor Wortmannin. The fraction of Akt phosphorylated at S473 was measured in cells stimulated for 30 min with 1 μg/ml ephrin-A1 Fc or Fc as a control and in cells treated with 20 nM Wortmannin for 30 min or left untreated (−) using the MesoScale technology. The histogram shows averages ± SD from triplicate measurements for ephrin-A1 and duplicate measurements for Wortmannin. (B) Ephrin-A1 Fc stimulation similarly inhibits Akt phosphorylation in the presence and in the absence of FBS. Cells grown in 10% FBS or starved overnight without serum were stimulated for 15 min with 1 μg/ml ephrin-A1 Fc or Fc as a control. Lysates were probed with the indicated antibodies. (C) EphA2 causes Akt dephosphorylation independently of PHLPP phosphatases. Cells were transfected with control siRNA or siRNAs targeting both PHLPP1 and PHLPP2 phosphatases and stimulated for 15 min with 0.1 μg/ml ephrin-A1 Fc or Fc as a control. Lysates were probed with the indicated antibodies. (D) Calyculin prevents ephrin-A1-dependent Akt dephosphorylation. PC3 cells were incubated for 30 min with the indicated concentrations of the phosphatase inhibitor calyculin, which similarly inhibits both PP1 and PP2A phosphatases. The cells were then stimulated for 15 min with with 1 mg/ml ephrin-A1 Fc or Fc as a control. Lysates were probed with the indicated antibodies. Blotting with anti-phosphothreonine antibodies (PThr) demonstrates overall serine/threonine phosphatase inactivation. (E) Tautomycin prevents ephrin-A1-dependent Akt dephosphorylation. PC3 cells were incubated for 4 hours with the phosphatase inhibitor tautomycin, which preferentially inhibits PP1 over PP2A. The cells were stimulated and analyzed as in (D). (F) Okadaic acid prevents ephrin-A1-dependent Akt dephosphorylation only when used at high concentrations. PC3 cells were incubated for 4 hours with the phosphatase inhibitor okadaic acid, which preferentially inhibits PP2A over PP1. The cells were stimulated and analyzed as in (D).
Figure Legend Snippet: Serine/threonine phosphatase activity is required for EphA2-dependent Akt dephosphorylation. (A) Ephrin-A1 decreases Akt phosphorylation almost as much as the potent PI3 kinase inhibitor Wortmannin. The fraction of Akt phosphorylated at S473 was measured in cells stimulated for 30 min with 1 μg/ml ephrin-A1 Fc or Fc as a control and in cells treated with 20 nM Wortmannin for 30 min or left untreated (−) using the MesoScale technology. The histogram shows averages ± SD from triplicate measurements for ephrin-A1 and duplicate measurements for Wortmannin. (B) Ephrin-A1 Fc stimulation similarly inhibits Akt phosphorylation in the presence and in the absence of FBS. Cells grown in 10% FBS or starved overnight without serum were stimulated for 15 min with 1 μg/ml ephrin-A1 Fc or Fc as a control. Lysates were probed with the indicated antibodies. (C) EphA2 causes Akt dephosphorylation independently of PHLPP phosphatases. Cells were transfected with control siRNA or siRNAs targeting both PHLPP1 and PHLPP2 phosphatases and stimulated for 15 min with 0.1 μg/ml ephrin-A1 Fc or Fc as a control. Lysates were probed with the indicated antibodies. (D) Calyculin prevents ephrin-A1-dependent Akt dephosphorylation. PC3 cells were incubated for 30 min with the indicated concentrations of the phosphatase inhibitor calyculin, which similarly inhibits both PP1 and PP2A phosphatases. The cells were then stimulated for 15 min with with 1 mg/ml ephrin-A1 Fc or Fc as a control. Lysates were probed with the indicated antibodies. Blotting with anti-phosphothreonine antibodies (PThr) demonstrates overall serine/threonine phosphatase inactivation. (E) Tautomycin prevents ephrin-A1-dependent Akt dephosphorylation. PC3 cells were incubated for 4 hours with the phosphatase inhibitor tautomycin, which preferentially inhibits PP1 over PP2A. The cells were stimulated and analyzed as in (D). (F) Okadaic acid prevents ephrin-A1-dependent Akt dephosphorylation only when used at high concentrations. PC3 cells were incubated for 4 hours with the phosphatase inhibitor okadaic acid, which preferentially inhibits PP2A over PP1. The cells were stimulated and analyzed as in (D).

Techniques Used: Activity Assay, De-Phosphorylation Assay, Transfection, Incubation

13) Product Images from "TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity"

Article Title: TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031225

TBC1D3 increases signaling through the activation of insulin pathway. (A) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for the indicated times. Phosphorylation and protein levels of Akt were analyzed by Western blotting. ( Right panel ) Quantification data of Akt:S473 phosphorylation normalized to Akt protein levels (* p
Figure Legend Snippet: TBC1D3 increases signaling through the activation of insulin pathway. (A) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for the indicated times. Phosphorylation and protein levels of Akt were analyzed by Western blotting. ( Right panel ) Quantification data of Akt:S473 phosphorylation normalized to Akt protein levels (* p

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Western Blot

14) Product Images from "A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop"

Article Title: A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop

Journal: Oncogenesis

doi: 10.1038/oncsis.2013.4

Effect of TKIs on ERK and AKT phosphorylation in control and gefitinib-resistant HCC4006 and H1650 cells. ( a , b ) HCC4006 and H1650 control (DMSO) or gefitinib-resistant cells were treated for 2 h with inhibitors: Reversible EGFR inhibitor (lapatinib/gefitinib, 1 μℳ), irreversible EGFR inhibitor (BIBW2992, 0.1 μℳ), FGFR inhibitors (AZD8010, 0.3 μℳ, RO4383596, 1 μℳ) or Met inhibitor (crizotinib, 0.2 μℳ). Cell lysates were immunoblotted for phospho-ERK and phospho-AKT-T308 and phospho-AKT-S473 as indicated. The filters were stripped and reprobed for total ERK1/2 and AKT as loading controls. The phospho-AKT-T308 blot was submitted to densitometry and a graphical presentation is shown in Supplementary Figure S4 .
Figure Legend Snippet: Effect of TKIs on ERK and AKT phosphorylation in control and gefitinib-resistant HCC4006 and H1650 cells. ( a , b ) HCC4006 and H1650 control (DMSO) or gefitinib-resistant cells were treated for 2 h with inhibitors: Reversible EGFR inhibitor (lapatinib/gefitinib, 1 μℳ), irreversible EGFR inhibitor (BIBW2992, 0.1 μℳ), FGFR inhibitors (AZD8010, 0.3 μℳ, RO4383596, 1 μℳ) or Met inhibitor (crizotinib, 0.2 μℳ). Cell lysates were immunoblotted for phospho-ERK and phospho-AKT-T308 and phospho-AKT-S473 as indicated. The filters were stripped and reprobed for total ERK1/2 and AKT as loading controls. The phospho-AKT-T308 blot was submitted to densitometry and a graphical presentation is shown in Supplementary Figure S4 .

Techniques Used:

15) Product Images from "Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system"

Article Title: Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system

Journal: Aging Cell

doi: 10.1111/acel.12405

Sustained impact of intermittent rapamycin on adipose m TORC 1 and testes weight. (A) Muscle lysate and (B) adipose tissue lysate were analyzed by Western blotting, and the phosphorylation of S6 240/244 and AKT S473 relative to their respective total protein was quantified. Tissues were collected from mice treated with vehicle or rapamycin (1×/day or 1×/5 days) for 8 weeks, with the tissue collection scheduled such that the intermittent rapamycin treatment group was sacrificed 5 days after the previous rapamycin injection. Mice were fasted overnight and sacrificed following stimulation with 0.75 U/kg insulin for 15 min. Islets were isolated as described prior to tissue collection [ n = 5–9 per treatment, means with the same letter are not significantly different from each other (Tukey–Kramer test following one‐way anova , P
Figure Legend Snippet: Sustained impact of intermittent rapamycin on adipose m TORC 1 and testes weight. (A) Muscle lysate and (B) adipose tissue lysate were analyzed by Western blotting, and the phosphorylation of S6 240/244 and AKT S473 relative to their respective total protein was quantified. Tissues were collected from mice treated with vehicle or rapamycin (1×/day or 1×/5 days) for 8 weeks, with the tissue collection scheduled such that the intermittent rapamycin treatment group was sacrificed 5 days after the previous rapamycin injection. Mice were fasted overnight and sacrificed following stimulation with 0.75 U/kg insulin for 15 min. Islets were isolated as described prior to tissue collection [ n = 5–9 per treatment, means with the same letter are not significantly different from each other (Tukey–Kramer test following one‐way anova , P

Techniques Used: Western Blot, Mouse Assay, Injection, Isolation

16) Product Images from "Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle"

Article Title: Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddp271

mTORC2 regulates cell size and cell cycle via a mechanism involving TSC2. ( A ) Simplified schematic presentation of the potential functional interaction of mTORC2 and mTORC1 in cell size regulation. Both, PI3K and mTORC2 (mTOR/rictor) are necessary to drive full activation of the serine-threonine kinase Akt, which in turn negatively regulates PRAS40 and the TSC1/2 complex to activate mTORC1 (mTOR/raptor) known to exert positive effects on cell size regulation via its effector S6K. (B–F) Logarithmically growing IMR-90 fibroblasts were either transfected with rictor siRNA alone or were co-transfected with siRNAs targeting rictor and PRAS40 or rictor and TSC2. Non-targeting siRNA (control) was analysed in parallel. Sixty hours post transfection, cells were replated into fresh growth medium and incubated for additional 20 h. For experiments presented in (D), cells were transfected as described above but were grown for a total of 96 h without replating. ( B ) siRNA-treated cells were harvested, lysed and protein levels of rictor, PRAS40 and tuberin were examined via immunoblotting to prove efficient knockdown of target proteins. In addition, the expression level of Akt and p70S6K as well as the corresponding phosphorylation status at S473 and T389, respectively, were analysed. Lysates of non-treated cells, which were co-analysed in parallel, were included to prove the here used conditions of siRNA treatment to be physiologic and technically sound (compare control versus non-treated). ( C ) siRNA-treated cells derived from the same pool of cells described in (B) were stained with propidium iodide and cytofluorometrically analysed for cell cycle distribution. Representative DNA profiles (upper panel) and the percentage of cells in G0/G1, S and G2/M (lower panel) are presented. ( D ) Lysates of cells treated as indicated were examined for the expression levels of rictor, tuberin, p27 and α-tubulin. In addition, cells were examined for overall cell size via FSC analyses ( E ) and for cell size according to different cell cycle phases via two-dimensional blots of FSC versus DNA-content ( F ) on the flow cytometer.
Figure Legend Snippet: mTORC2 regulates cell size and cell cycle via a mechanism involving TSC2. ( A ) Simplified schematic presentation of the potential functional interaction of mTORC2 and mTORC1 in cell size regulation. Both, PI3K and mTORC2 (mTOR/rictor) are necessary to drive full activation of the serine-threonine kinase Akt, which in turn negatively regulates PRAS40 and the TSC1/2 complex to activate mTORC1 (mTOR/raptor) known to exert positive effects on cell size regulation via its effector S6K. (B–F) Logarithmically growing IMR-90 fibroblasts were either transfected with rictor siRNA alone or were co-transfected with siRNAs targeting rictor and PRAS40 or rictor and TSC2. Non-targeting siRNA (control) was analysed in parallel. Sixty hours post transfection, cells were replated into fresh growth medium and incubated for additional 20 h. For experiments presented in (D), cells were transfected as described above but were grown for a total of 96 h without replating. ( B ) siRNA-treated cells were harvested, lysed and protein levels of rictor, PRAS40 and tuberin were examined via immunoblotting to prove efficient knockdown of target proteins. In addition, the expression level of Akt and p70S6K as well as the corresponding phosphorylation status at S473 and T389, respectively, were analysed. Lysates of non-treated cells, which were co-analysed in parallel, were included to prove the here used conditions of siRNA treatment to be physiologic and technically sound (compare control versus non-treated). ( C ) siRNA-treated cells derived from the same pool of cells described in (B) were stained with propidium iodide and cytofluorometrically analysed for cell cycle distribution. Representative DNA profiles (upper panel) and the percentage of cells in G0/G1, S and G2/M (lower panel) are presented. ( D ) Lysates of cells treated as indicated were examined for the expression levels of rictor, tuberin, p27 and α-tubulin. In addition, cells were examined for overall cell size via FSC analyses ( E ) and for cell size according to different cell cycle phases via two-dimensional blots of FSC versus DNA-content ( F ) on the flow cytometer.

Techniques Used: Functional Assay, Activation Assay, Transfection, Incubation, Expressing, Derivative Assay, Staining, Flow Cytometry, Cytometry

Blocking mTORC1 activity delays S-phase induction without effects on mTORC2 activity. IMR-90 fibroblasts, synchronized in G0/G1 via serum deprivation, were serum restimulated to re-enter the cell cycle in the presence or absence of the mTOR inhibitor rapamycin. ( A ) Schematic outline of the synchronization procedure. Briefly, cells growing under full serum (10% FCS) conditions for 24–72 h were serum-deprived in medium containing 0.2% serum (0.2% FCS) for 48 h and serum restimulated (10% FCS) for additional 36 h. Rapamycin was added 30 min prior to restimulation at a concentration of 100 n m . Control cells were treated with an equal volume of DMSO and analysed in parallel. ( B ) Cells treated as described in (A) were lysed and examined for the expression level of cyclin D1, cyclin A, p70S6K T389, p70S6K, Akt S473 and Akt at the indicated time points via immunoblotting. ( C ) In addition, cells were cytofluorometrically analysed for DNA content at the indicated time points. DNA profiles are presented (upper panel). In addition, the percentage of cells in S-phase was determined for DMSO (vehicle)- and rapamycin-treated cells. To facilitate interpretation of S-phase induction, a baseline corresponding to the S-phase values of serum-deprived vehicle- and rapamycin-treated cells was inserted into the graph. ( D ) Experiments as described in (A, B and C) were repeated and the percentage of cells actively undergoing DNA replication was analysed by immunocytochemical BrdU incorporation assay at specific time points as indicated (at least 250 cells were scored for each timepoint). ( E ) In addition, cells derived from the same pool of cells as described in (D) were immunocytocemically stained for cyclin A expression (at least 250 cells were scored for each timepoint).
Figure Legend Snippet: Blocking mTORC1 activity delays S-phase induction without effects on mTORC2 activity. IMR-90 fibroblasts, synchronized in G0/G1 via serum deprivation, were serum restimulated to re-enter the cell cycle in the presence or absence of the mTOR inhibitor rapamycin. ( A ) Schematic outline of the synchronization procedure. Briefly, cells growing under full serum (10% FCS) conditions for 24–72 h were serum-deprived in medium containing 0.2% serum (0.2% FCS) for 48 h and serum restimulated (10% FCS) for additional 36 h. Rapamycin was added 30 min prior to restimulation at a concentration of 100 n m . Control cells were treated with an equal volume of DMSO and analysed in parallel. ( B ) Cells treated as described in (A) were lysed and examined for the expression level of cyclin D1, cyclin A, p70S6K T389, p70S6K, Akt S473 and Akt at the indicated time points via immunoblotting. ( C ) In addition, cells were cytofluorometrically analysed for DNA content at the indicated time points. DNA profiles are presented (upper panel). In addition, the percentage of cells in S-phase was determined for DMSO (vehicle)- and rapamycin-treated cells. To facilitate interpretation of S-phase induction, a baseline corresponding to the S-phase values of serum-deprived vehicle- and rapamycin-treated cells was inserted into the graph. ( D ) Experiments as described in (A, B and C) were repeated and the percentage of cells actively undergoing DNA replication was analysed by immunocytochemical BrdU incorporation assay at specific time points as indicated (at least 250 cells were scored for each timepoint). ( E ) In addition, cells derived from the same pool of cells as described in (D) were immunocytocemically stained for cyclin A expression (at least 250 cells were scored for each timepoint).

Techniques Used: Blocking Assay, Activity Assay, Concentration Assay, Expressing, BrdU Incorporation Assay, Derivative Assay, Staining

mTORC2 regulates mammalian cell size and cell cycle progression. Logarithmically growing IMR-90 fibroblasts were transfected with short-interfering RNAs (siRNAs) targeting human raptor and rictor, respectively. Cells treated with non-targeting siRNA were analysed in parallel and served as a negative control (control). Forty-eight hours after transfection, cells were replated at low density and were grown for another 20 h. ( A ) Cells as described above were lysed and examined for the expression level of raptor, rictor, S473 phosphorylated Akt, total Akt, T389 phosphorylated p70S6K and total p70S6K. α-Tubulin was co-analysed as an additional loading control. ( B ) Cells derived from the same pool of cells described in (A) were cytofluorometrically analysed for DNA distribution. Representative DNA profiles (upper panel) and the percentage of cells in G0/G1, S and G2/M (lower panel) are presented. Apart from the quantification of DNA distribution, cells were cytofluorometrically examined for overall cell size via FSC analyses ( C ) and for cell size according to different cell cycle phases via two-dimensional blots of FSC versus DNA content ( D ).
Figure Legend Snippet: mTORC2 regulates mammalian cell size and cell cycle progression. Logarithmically growing IMR-90 fibroblasts were transfected with short-interfering RNAs (siRNAs) targeting human raptor and rictor, respectively. Cells treated with non-targeting siRNA were analysed in parallel and served as a negative control (control). Forty-eight hours after transfection, cells were replated at low density and were grown for another 20 h. ( A ) Cells as described above were lysed and examined for the expression level of raptor, rictor, S473 phosphorylated Akt, total Akt, T389 phosphorylated p70S6K and total p70S6K. α-Tubulin was co-analysed as an additional loading control. ( B ) Cells derived from the same pool of cells described in (A) were cytofluorometrically analysed for DNA distribution. Representative DNA profiles (upper panel) and the percentage of cells in G0/G1, S and G2/M (lower panel) are presented. Apart from the quantification of DNA distribution, cells were cytofluorometrically examined for overall cell size via FSC analyses ( C ) and for cell size according to different cell cycle phases via two-dimensional blots of FSC versus DNA content ( D ).

Techniques Used: Transfection, Negative Control, Expressing, Derivative Assay

mTORC1-mediated consequences on cell cycle and cell size are separable and do not involve effects on mTORC2 activity. ( A ) Logarithmically growing non-transformed, non-immortalized IMR-90 fibroblasts, derived from human fetal lung tissue, were treated with the mTOR inhibitor rapamycin at final concentrations ranging from 0.1 n m to 100 n m for 24 h. Control cells were treated with an equal volume of DMSO (vehicle). Total lysates were prepared and analysed for the expression level of p70S6K and Akt as well as for the phosphorylation status at T389 and S473, respectively. ( B ) In addition, so treated cells were stained with propidium iodide and cytofluorometrically analysed for DNA distribution. Representative DNA profiles are presented (upper panel). The percentage of cells in G0/G1, S and G2/M is indicated (lower panel). ( C ) Phospho-Akt S473 signals detected in (A) were densitometrically scanned and normalized to total Akt levels. ( D ) 100 n m rapamycin-treated IMR-90 cells derived from the same pool of cells as analysed in (A, B and C) were cytofluorometrically investigated for overall cell size via FSC (Forward Scatter) analyses. Overlays of FSC histograms are presented to enable direct comparison of control cells versus rapamycin-treated cells (left panel). Mean FSC values obtained from these analyses are presented (right panel). ( E ) In addition, cell size according to different cell cycle phases was examined via two-dimensional contour blot analyses of FSC versus DNA content. To visualize FSC shifts between different samples, a crosshair has been set into the center of the G0/G1 population of control cells (left panel). For details regarding different types of FSC analyses compare the Methods section in the text. Furthermore, G0/G1-phase cells (2 N) and G2/M-phase cells (4 N) were selectively gated and analysed for cell size distribution via FSC. Mean FSC values and the percentage of gated cells are presented (right panel). ( F ) Lysates of IMR-90 cells treated with 100 n m rapamycin for the indicated times were analysed for the expression level of total p70S6K (on long exposures the used α-p70S6K antibody recognizes also lower migrating p85S6K) and total Akt as well as for their phosphorylated forms as indicated. In addition, same lysates were examined for cyclin A levels. ( G ) Logarithmically growing IMR-90 cells (vehicle) were either treated with 100 n m rapamycin for 24 h as described in (A) or were grown under low proliferation conditions. So treated cells were cytofluorometrically analysed for DNA distribution. The percentage of cells in S-phase is indicated. ( H ) In parallel, overall cell size was cytofluorometrically investigated via FSC (upper panel). Mean FSC values obtained from these analyses are presented (lower panel). ( I ) In addition, cell size according to different cell cycle phases was examined via two-dimensional contour blot analyses of FSC versus DNA content. ( J ) IMR-90 fibroblasts were treated with 100 n m rapamycin for the indicated times and total cell lysates were analysed for the expression levels of p70S6K T389, total p70S6K, Akt S473, total Akt, cyclin A and cyclin D1. In addition, the percentage of cells in S-phase was determined on the flow cytometer. ( K ) Phospho-Akt S473 signals detected in (J) were densitometrically scanned and normalized to total Akt levels. ( L ) In parallel, overall cell size distribution of so treated cells was cytofluorometrically analysed via FSC at the indicated time points. ( M ) Logarithmically growing IMR-90 fibroblasts were transfected with pooled short-interfering RNAs (siRNAs) targeting human rictor (si rictor). Cells treated with non-targeting siRNAs (control) or cells which were left entirely untreated were analysed in parallel. About 72 h after initial transfection, lysates were prepared and analysed for indicated proteins via immunoblotting. ( N ) Experiments were basically performed as described in (M) with the exception that cells were treated with 100 n m rapamycin or DMSO for the final 24 h of incubation. So treated cells were lysed and examined for the expression level of rictor, S473 phosphorylated Akt, total Akt, T389 phosphorylated p70S6K and total p70S6K.
Figure Legend Snippet: mTORC1-mediated consequences on cell cycle and cell size are separable and do not involve effects on mTORC2 activity. ( A ) Logarithmically growing non-transformed, non-immortalized IMR-90 fibroblasts, derived from human fetal lung tissue, were treated with the mTOR inhibitor rapamycin at final concentrations ranging from 0.1 n m to 100 n m for 24 h. Control cells were treated with an equal volume of DMSO (vehicle). Total lysates were prepared and analysed for the expression level of p70S6K and Akt as well as for the phosphorylation status at T389 and S473, respectively. ( B ) In addition, so treated cells were stained with propidium iodide and cytofluorometrically analysed for DNA distribution. Representative DNA profiles are presented (upper panel). The percentage of cells in G0/G1, S and G2/M is indicated (lower panel). ( C ) Phospho-Akt S473 signals detected in (A) were densitometrically scanned and normalized to total Akt levels. ( D ) 100 n m rapamycin-treated IMR-90 cells derived from the same pool of cells as analysed in (A, B and C) were cytofluorometrically investigated for overall cell size via FSC (Forward Scatter) analyses. Overlays of FSC histograms are presented to enable direct comparison of control cells versus rapamycin-treated cells (left panel). Mean FSC values obtained from these analyses are presented (right panel). ( E ) In addition, cell size according to different cell cycle phases was examined via two-dimensional contour blot analyses of FSC versus DNA content. To visualize FSC shifts between different samples, a crosshair has been set into the center of the G0/G1 population of control cells (left panel). For details regarding different types of FSC analyses compare the Methods section in the text. Furthermore, G0/G1-phase cells (2 N) and G2/M-phase cells (4 N) were selectively gated and analysed for cell size distribution via FSC. Mean FSC values and the percentage of gated cells are presented (right panel). ( F ) Lysates of IMR-90 cells treated with 100 n m rapamycin for the indicated times were analysed for the expression level of total p70S6K (on long exposures the used α-p70S6K antibody recognizes also lower migrating p85S6K) and total Akt as well as for their phosphorylated forms as indicated. In addition, same lysates were examined for cyclin A levels. ( G ) Logarithmically growing IMR-90 cells (vehicle) were either treated with 100 n m rapamycin for 24 h as described in (A) or were grown under low proliferation conditions. So treated cells were cytofluorometrically analysed for DNA distribution. The percentage of cells in S-phase is indicated. ( H ) In parallel, overall cell size was cytofluorometrically investigated via FSC (upper panel). Mean FSC values obtained from these analyses are presented (lower panel). ( I ) In addition, cell size according to different cell cycle phases was examined via two-dimensional contour blot analyses of FSC versus DNA content. ( J ) IMR-90 fibroblasts were treated with 100 n m rapamycin for the indicated times and total cell lysates were analysed for the expression levels of p70S6K T389, total p70S6K, Akt S473, total Akt, cyclin A and cyclin D1. In addition, the percentage of cells in S-phase was determined on the flow cytometer. ( K ) Phospho-Akt S473 signals detected in (J) were densitometrically scanned and normalized to total Akt levels. ( L ) In parallel, overall cell size distribution of so treated cells was cytofluorometrically analysed via FSC at the indicated time points. ( M ) Logarithmically growing IMR-90 fibroblasts were transfected with pooled short-interfering RNAs (siRNAs) targeting human rictor (si rictor). Cells treated with non-targeting siRNAs (control) or cells which were left entirely untreated were analysed in parallel. About 72 h after initial transfection, lysates were prepared and analysed for indicated proteins via immunoblotting. ( N ) Experiments were basically performed as described in (M) with the exception that cells were treated with 100 n m rapamycin or DMSO for the final 24 h of incubation. So treated cells were lysed and examined for the expression level of rictor, S473 phosphorylated Akt, total Akt, T389 phosphorylated p70S6K and total p70S6K.

Techniques Used: Activity Assay, Transformation Assay, Derivative Assay, Expressing, Staining, Flow Cytometry, Cytometry, Transfection, Incubation

17) Product Images from "NHERF1/EBP50 controls lactation by establishing basal membrane polarity complexes with prolactin receptor"

Article Title: NHERF1/EBP50 controls lactation by establishing basal membrane polarity complexes with prolactin receptor

Journal: Cell Death & Disease

doi: 10.1038/cddis.2012.131

NHERF1 loss increases Akt activation and proliferation in mammary ducts. ( a ) Western blot of mammary gland lysates as in Figure 2c . The graphs show the densitometric intensities of phosphorylated S473 Akt levels normalized to total Akt levels. n =2 blots. ( b ) BrdU staining of mammary gland in 6-week-old NHERF1 (+/+) and (−/−) virgin littermates. The graph shows the normalized BrdU-positive cell count per 0.2-mm 2 mammary duct area. n =35 ducts
Figure Legend Snippet: NHERF1 loss increases Akt activation and proliferation in mammary ducts. ( a ) Western blot of mammary gland lysates as in Figure 2c . The graphs show the densitometric intensities of phosphorylated S473 Akt levels normalized to total Akt levels. n =2 blots. ( b ) BrdU staining of mammary gland in 6-week-old NHERF1 (+/+) and (−/−) virgin littermates. The graph shows the normalized BrdU-positive cell count per 0.2-mm 2 mammary duct area. n =35 ducts

Techniques Used: Activation Assay, Western Blot, BrdU Staining, Cell Counting

18) Product Images from "Targeting promiscuous heterodimerization overcomes innate resistance to ERBB2 dimerization inhibitors in breast cancer"

Article Title: Targeting promiscuous heterodimerization overcomes innate resistance to ERBB2 dimerization inhibitors in breast cancer

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-019-1127-y

Targeting ERBB2 with therapeutic monoclonal antibodies. a Western blotting showing the expression of ERBB2, pAkt S473 and Akt in a panel of breast cancer cell lines. b Cell viability assays performed on the panel of lines with trastuzumab and pertuzumab at the concentrations indicated for 5 days ( n = 6, mean ± SD). c Combinatorial cell viability assays with trastuzumab and pertuzumab at the concentrations indicated for 5 days ( n = 6, mean). Raw data is presented in Additional file 2 : Figure S1B. d Western blotting showing the phosphorylation of ERBB3 Y1289 and Akt S473 following treatment with trastuzumab (100 nM), pertuzumab (100 nM) and the combination of both, at the time points indicated. Blots were re-probed with an actin antibody for quantification. A representative image is shown from three independent replicates ( n = 3, mean ± SD). All drug treatments were significantly different from control. For simplicity, the indicated significance compares the combination to trastuzumab only (* p
Figure Legend Snippet: Targeting ERBB2 with therapeutic monoclonal antibodies. a Western blotting showing the expression of ERBB2, pAkt S473 and Akt in a panel of breast cancer cell lines. b Cell viability assays performed on the panel of lines with trastuzumab and pertuzumab at the concentrations indicated for 5 days ( n = 6, mean ± SD). c Combinatorial cell viability assays with trastuzumab and pertuzumab at the concentrations indicated for 5 days ( n = 6, mean). Raw data is presented in Additional file 2 : Figure S1B. d Western blotting showing the phosphorylation of ERBB3 Y1289 and Akt S473 following treatment with trastuzumab (100 nM), pertuzumab (100 nM) and the combination of both, at the time points indicated. Blots were re-probed with an actin antibody for quantification. A representative image is shown from three independent replicates ( n = 3, mean ± SD). All drug treatments were significantly different from control. For simplicity, the indicated significance compares the combination to trastuzumab only (* p

Techniques Used: Western Blot, Expressing

19) Product Images from "Histone Demethylase KDM4C Stimulates the Proliferation of Prostate Cancer Cells via Activation of AKT and c-Myc"

Article Title: Histone Demethylase KDM4C Stimulates the Proliferation of Prostate Cancer Cells via Activation of AKT and c-Myc

Journal: Cancers

doi: 10.3390/cancers11111785

Micro-Western array (MWA) and Western blotting analysis indicated that knockdown of KDM4C suppressed AKT signaling and c-Myc in PCa cells. ( A ) Expression of proteins involved in cell cycle regulation, EGFR signaling, AKT signaling, Src signaling, and JNK signaling in LNCaP C4-2B cells with or without KDM4C siRNA KD was analyzed with MWA. ( B ) Expression level of proteins with a fold change larger than 2.0 or smaller than 0.5 between control and KDM4C knockdown analyzed from ( A ) is demonstrated in a heatmap with the value of the protein expression level being converted to log 2 . ( C ) Western blotting assay was used to confirm the change in proteins observed by MWA. Expression of KDM4C, mTOR, PTEN, PDK1, phospho-PDK1 S241, c-Myc, AKT, phospho-AKT T308 and phospho-AKT S473 was determined in triplicates of control LNCaP C4-2B cells and LNCaP C4-2B cells with KDM4C knockdown. β-Actin was used as the loading control.
Figure Legend Snippet: Micro-Western array (MWA) and Western blotting analysis indicated that knockdown of KDM4C suppressed AKT signaling and c-Myc in PCa cells. ( A ) Expression of proteins involved in cell cycle regulation, EGFR signaling, AKT signaling, Src signaling, and JNK signaling in LNCaP C4-2B cells with or without KDM4C siRNA KD was analyzed with MWA. ( B ) Expression level of proteins with a fold change larger than 2.0 or smaller than 0.5 between control and KDM4C knockdown analyzed from ( A ) is demonstrated in a heatmap with the value of the protein expression level being converted to log 2 . ( C ) Western blotting assay was used to confirm the change in proteins observed by MWA. Expression of KDM4C, mTOR, PTEN, PDK1, phospho-PDK1 S241, c-Myc, AKT, phospho-AKT T308 and phospho-AKT S473 was determined in triplicates of control LNCaP C4-2B cells and LNCaP C4-2B cells with KDM4C knockdown. β-Actin was used as the loading control.

Techniques Used: Western Blot, Expressing

20) Product Images from "Expression of the RAE-1 Family of Stimulatory NK-Cell Ligands Requires Activation of the PI3K Pathway during Viral Infection and Transformation"

Article Title: Expression of the RAE-1 Family of Stimulatory NK-Cell Ligands Requires Activation of the PI3K Pathway during Viral Infection and Transformation

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002265

PI3K pathway is required for RAE-1 induction in MCMV-infected cells through the activation of Akt. A ) Fibroblasts infected with MCMVΔ152 in the presence of 20 uM LY294002 were stained for RAE-1 at 24 hrs pi. Histograms show isotype control (shaded gray), uninfected (solid gray), MCMVΔ152-infected (solid black), and MCMVΔ152-infected cells in the presence of LY294002 (dashed black). The histogram is a representative figure from three independent experiments. B ) Plaque forming units (PFU)/ml were determined in supernatants of infected cells in the presence of DMSO or LY294002. SD and statistical significance were determined from three independent experiments. NS, not statistically significant. C ) Whole cell lysates from fibroblasts infected with MCMV WT in the presence of DMSO or LY294002 were obtained at 12 and 24 hrs pi. Lysates were also obtained from cells treated with 20% FBS or cells overexpressing p110α H1047R. All samples were blotted for phospho-Akt S473 and total Akt. The relative ratio of P-Akt to total Akt from the blot was determined. The data is a representative experiment from ten independent experiments.
Figure Legend Snippet: PI3K pathway is required for RAE-1 induction in MCMV-infected cells through the activation of Akt. A ) Fibroblasts infected with MCMVΔ152 in the presence of 20 uM LY294002 were stained for RAE-1 at 24 hrs pi. Histograms show isotype control (shaded gray), uninfected (solid gray), MCMVΔ152-infected (solid black), and MCMVΔ152-infected cells in the presence of LY294002 (dashed black). The histogram is a representative figure from three independent experiments. B ) Plaque forming units (PFU)/ml were determined in supernatants of infected cells in the presence of DMSO or LY294002. SD and statistical significance were determined from three independent experiments. NS, not statistically significant. C ) Whole cell lysates from fibroblasts infected with MCMV WT in the presence of DMSO or LY294002 were obtained at 12 and 24 hrs pi. Lysates were also obtained from cells treated with 20% FBS or cells overexpressing p110α H1047R. All samples were blotted for phospho-Akt S473 and total Akt. The relative ratio of P-Akt to total Akt from the blot was determined. The data is a representative experiment from ten independent experiments.

Techniques Used: Infection, Activation Assay, Staining

21) Product Images from "A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration"

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

Journal: eLife

doi: 10.7554/eLife.36645

Human phospho-kinase array studies of TYPE7 specificity. H358 cells were treated for 10 min with TYPE7 (2 μM) and the following controls: Fc (CT), EA1 (0.5 μg/mL) and pHLIP (2 μM). After treatment, cell lysates were incubated overnight with array membranes (R and D Systems ARY003B) for duplicated detection of phosphorylation of 43 total kinases ( A ) and their substrates ( B ). Myristoylated Src family kinases are boxed: top (Hck, Fyn and Src), middle (Yes and Lyn), and bottom (Lck). The pHLIP peptide was used as a control for specificity. The array contains the following proteins, in order from top to bottom, and then left to right: p38α, ERK1/2, JNK 1/2/3, GSK-3α/β, p53, EGFR, MSK1/2, AMPKα1, Akt, p53, TOR, CREB, HSP27, AMPKα2, β-Catenin, p70 S6 Kinase, p53, c-Jun, Src, Lyn, Lck, STAT2, STAT5a, p70 S6 Kinase, RSK1/2/3, eNOS, Fyn, Yes, Fgr, STAT6, STAT5b, STAT3, p27, PLC-g1, Hck, Chk-2, FAX, PDGFRb, STAT5a/b, STAT3, WNK1, PYK2, PRAS40 and HSP60. ( C ) Quantification of Akt phosphorylation (p–S473).
Figure Legend Snippet: Human phospho-kinase array studies of TYPE7 specificity. H358 cells were treated for 10 min with TYPE7 (2 μM) and the following controls: Fc (CT), EA1 (0.5 μg/mL) and pHLIP (2 μM). After treatment, cell lysates were incubated overnight with array membranes (R and D Systems ARY003B) for duplicated detection of phosphorylation of 43 total kinases ( A ) and their substrates ( B ). Myristoylated Src family kinases are boxed: top (Hck, Fyn and Src), middle (Yes and Lyn), and bottom (Lck). The pHLIP peptide was used as a control for specificity. The array contains the following proteins, in order from top to bottom, and then left to right: p38α, ERK1/2, JNK 1/2/3, GSK-3α/β, p53, EGFR, MSK1/2, AMPKα1, Akt, p53, TOR, CREB, HSP27, AMPKα2, β-Catenin, p70 S6 Kinase, p53, c-Jun, Src, Lyn, Lck, STAT2, STAT5a, p70 S6 Kinase, RSK1/2/3, eNOS, Fyn, Yes, Fgr, STAT6, STAT5b, STAT3, p27, PLC-g1, Hck, Chk-2, FAX, PDGFRb, STAT5a/b, STAT3, WNK1, PYK2, PRAS40 and HSP60. ( C ) Quantification of Akt phosphorylation (p–S473).

Techniques Used: Incubation, Planar Chromatography

22) Product Images from "AKT3 promotes prostate cancer proliferation cells through regulation of Akt, B-Raf TSC1/TSC2"

Article Title: AKT3 promotes prostate cancer proliferation cells through regulation of Akt, B-Raf TSC1/TSC2

Journal: Oncotarget

doi:

AKT3 overexpression and siRNA knockdown affected expression of signaling proteins in PC-3 cells Protein expression of total AKT, AKT3, phospho-AKT S473, phospho-AKT T308, c-Myc, Skp2, p21 Cip , p27 Kip , cyclin D1, cyclin E, GSK3α, phospho-GSK-3α S21, GSK3β, phospho-GSK-3β S9, B-Raf, TSC1, TSC2, mTOR, phosphor-mTOR S2448, p70S6K, phopsho-p70S6K T421/S424 in PC-3 vector control, PC-3 overexpressing AKT3, PC-3 scramble control, and PC-3 AKT3 siRNA knockdown was assayed by Western blotting. Protein abundance of α-tubulin and β-actin were used as loading control.
Figure Legend Snippet: AKT3 overexpression and siRNA knockdown affected expression of signaling proteins in PC-3 cells Protein expression of total AKT, AKT3, phospho-AKT S473, phospho-AKT T308, c-Myc, Skp2, p21 Cip , p27 Kip , cyclin D1, cyclin E, GSK3α, phospho-GSK-3α S21, GSK3β, phospho-GSK-3β S9, B-Raf, TSC1, TSC2, mTOR, phosphor-mTOR S2448, p70S6K, phopsho-p70S6K T421/S424 in PC-3 vector control, PC-3 overexpressing AKT3, PC-3 scramble control, and PC-3 AKT3 siRNA knockdown was assayed by Western blotting. Protein abundance of α-tubulin and β-actin were used as loading control.

Techniques Used: Over Expression, Expressing, Plasmid Preparation, Western Blot

23) Product Images from "Phosphorylation of the Androgen Receptor by PIM1 in Hormone Refractory Prostate Cancer"

Article Title: Phosphorylation of the Androgen Receptor by PIM1 in Hormone Refractory Prostate Cancer

Journal: Oncogene

doi: 10.1038/onc.2012.412

PIM1 Phosphorylates AR in vitro A 293 cells were transiently transfected with FLAG-AR. Cells were steroid starved overnight and then treated with vehicle or 10nM R1881 for 2 hours. FLAG-AR was immunopurified and then subjected to kinase reactions in the presence of recombinant GSK3, PIM1, or Akt. The proteins were immunoblotted for P-AR S213, AR, PIM1, and P-Akt S473. B and C Kinase reactions using recombinant His-Akt or GST-PIM1 with their known substrates, GSK3 and Bad, respectively. Kinase activity was detected using antibodies against P-GSK3α/β S21/9, P-Akt S473, and P-Bad S112. D Recombinant AR and PIM1 were combined in a kinase reaction in the presence of R1881. Phosphorylation was detected by P-AR S213 antibody. AR and PIM1 antibodies were also used as controls for the presence of AR and PIM1.
Figure Legend Snippet: PIM1 Phosphorylates AR in vitro A 293 cells were transiently transfected with FLAG-AR. Cells were steroid starved overnight and then treated with vehicle or 10nM R1881 for 2 hours. FLAG-AR was immunopurified and then subjected to kinase reactions in the presence of recombinant GSK3, PIM1, or Akt. The proteins were immunoblotted for P-AR S213, AR, PIM1, and P-Akt S473. B and C Kinase reactions using recombinant His-Akt or GST-PIM1 with their known substrates, GSK3 and Bad, respectively. Kinase activity was detected using antibodies against P-GSK3α/β S21/9, P-Akt S473, and P-Bad S112. D Recombinant AR and PIM1 were combined in a kinase reaction in the presence of R1881. Phosphorylation was detected by P-AR S213 antibody. AR and PIM1 antibodies were also used as controls for the presence of AR and PIM1.

Techniques Used: In Vitro, Transfection, Recombinant, Activity Assay

24) Product Images from "Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning"

Article Title: Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200507119

AKT phosphorylation of tuberin promotes Rheb-induced S6K1 activation through increased Rheb-GTP loading. (A) HEK293 cells coexpressing HA-S6K1, Myc-Rheb, Flag-TSC1, and either Flag-TSC2-WT or Flag-TSC2-2A (S939A and S981A) were serum starved and, where indicated, the PI3K–AKT signaling pathway was stimulated with 100 nM insulin for 7.5 and 15 min. These cells were subjected to in vivo radiolabeling, and the level of guanine nucleotide bound to immunoprecipitated Myc-Rheb was quantified. A representative blot from three independent biological replications is shown. The mean of the percentage of total Myc-Rheb bound to GTP (active state) is shown in the bar figure. (B) In parallel, HEK293 cells treated as in A were subjected to S6K1 activity assays. HA-S6K1 used in the activity assay was analyzed with an anti-HA antibody. 32 P-incorporation into GST-S6 substrate was quantified using a phosphorimager, and the fold activation of S6K1 is graphed. Protein levels of Flag-TSC1, Flag-TSC2, and AKT, and level of AKT phosphorylation at S473, were determined using Western analyses.
Figure Legend Snippet: AKT phosphorylation of tuberin promotes Rheb-induced S6K1 activation through increased Rheb-GTP loading. (A) HEK293 cells coexpressing HA-S6K1, Myc-Rheb, Flag-TSC1, and either Flag-TSC2-WT or Flag-TSC2-2A (S939A and S981A) were serum starved and, where indicated, the PI3K–AKT signaling pathway was stimulated with 100 nM insulin for 7.5 and 15 min. These cells were subjected to in vivo radiolabeling, and the level of guanine nucleotide bound to immunoprecipitated Myc-Rheb was quantified. A representative blot from three independent biological replications is shown. The mean of the percentage of total Myc-Rheb bound to GTP (active state) is shown in the bar figure. (B) In parallel, HEK293 cells treated as in A were subjected to S6K1 activity assays. HA-S6K1 used in the activity assay was analyzed with an anti-HA antibody. 32 P-incorporation into GST-S6 substrate was quantified using a phosphorimager, and the fold activation of S6K1 is graphed. Protein levels of Flag-TSC1, Flag-TSC2, and AKT, and level of AKT phosphorylation at S473, were determined using Western analyses.

Techniques Used: Activation Assay, In Vivo, Radioactivity, Immunoprecipitation, Activity Assay, Western Blot

25) Product Images from "CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity"

Article Title: CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity

Journal: Scientific Reports

doi: 10.1038/s41598-017-17119-1

Generation of triple-deleted Trp53 −/− ; Brca2 −/− ; Pten −/− ID8 cells. ( A ) Immunoblot for PTEN and phospho-AKT following overnight serum starvation in clones isolated following Pten gRNA transfection. Clones 2.14.22 and 3.15.10, with bi-allelic Pten indels, showed absent PTEN expression and increased phosphorylation of AKT at both S473 and T308 compared to the two guide control clones, 2.14.10 and 3.15.7 that had no detectable change in Pten sequence. ( B ) ID8 Trp53 −/− and Trp53 −/− ; Brca2 −/− ; Pten −/− cells were irradiated (10 Gy), fixed and stained for γH2AX and RAD51, and counterstained with DAPI. RAD51 foci were counted in up to 30 untreated and irradiated cells. Bars represent foci per cell (mean +/− SEM); γH2AX (left) and RAD51 (right); dotted lines represent two-fold increase in γH2AX and RAD51 foci/cell relative to untreated cells as above.
Figure Legend Snippet: Generation of triple-deleted Trp53 −/− ; Brca2 −/− ; Pten −/− ID8 cells. ( A ) Immunoblot for PTEN and phospho-AKT following overnight serum starvation in clones isolated following Pten gRNA transfection. Clones 2.14.22 and 3.15.10, with bi-allelic Pten indels, showed absent PTEN expression and increased phosphorylation of AKT at both S473 and T308 compared to the two guide control clones, 2.14.10 and 3.15.7 that had no detectable change in Pten sequence. ( B ) ID8 Trp53 −/− and Trp53 −/− ; Brca2 −/− ; Pten −/− cells were irradiated (10 Gy), fixed and stained for γH2AX and RAD51, and counterstained with DAPI. RAD51 foci were counted in up to 30 untreated and irradiated cells. Bars represent foci per cell (mean +/− SEM); γH2AX (left) and RAD51 (right); dotted lines represent two-fold increase in γH2AX and RAD51 foci/cell relative to untreated cells as above.

Techniques Used: Clone Assay, Isolation, Transfection, Expressing, Sequencing, Irradiation, Staining

Generation of Trp53 −/− ; Nf1 −/− and Trp53 −/− ; Pten −/− ID8 cells. ( A ) Design of guide RNA targeted to exon 5 of Pten . Nucleotides in red represent PAM sequence. Schematic representation of PTEN protein with exon 5 highlighted in red (bottom). Numbers represent amino acid position. ( B ) Immunoblot for PTEN and phospho-AKT in clones following Pten gRNA transfection. Clones 1.11, 1.12, 1.14 and 1.15 had bi-allelic Pten deletions and showed absent PTEN protein with phosphorylation of AKT at both S473 and T308 following serum starvation. Clones 1.9 and 1.10 had mono-allelic deletions. F3 = ID8 Trp53 −/− . ( C ) Design of guide RNA targeted to exon 2 of Nf1 as previously 29 . Nucleotides in red represent PAM sequence. Schematic representation of Nf1 protein with exon 2 highlighted in red (bottom). Numbers represent amino acid positions. ( D ) Immunoblot for Nf1 in clones following Nf1 gRNA transfection. *; clones 1.20 and 1.23 had bi-allelic indels confirmed by Sanger sequencing, ^; clone 1.6 had single allele deletion. Clones 1.2, 1.3, 1.4, 1.7 and 1.17 were not sequenced. ( E ) Ras-GTP co-immunoprecipitation and phospho-ERK immunoblot in ID8 clones following Nf1 gRNA transfection. *; clones 1.20, 1.23, 1.30 and 1.56 had confirmed bi-allelic indels and demonstrated increased Ras-GTP pulldown and ERK phosphorylation suggestive of activated Ras signalling. ^; clones 1.6 and 1.58 had single allele deletions. ( F ) ID8 Trp53 −/− ; Pten −/− , Trp53 −/− ; Nf1 −/− and Trp53 −/− ; Pten + /− cells were irradiated (10 Gy), fixed and stained for γH2AX and RAD51, and counterstained with DAPI. RAD51 foci were counted in up to 30 untreated and irradiated cells. Bars represent foci per cell (mean +/− SEM); γH2AX (left) and RAD51 (right); dotted lines represents two-fold increase in γH2AX and RAD51 foci/cell relative to untreated cells as above.
Figure Legend Snippet: Generation of Trp53 −/− ; Nf1 −/− and Trp53 −/− ; Pten −/− ID8 cells. ( A ) Design of guide RNA targeted to exon 5 of Pten . Nucleotides in red represent PAM sequence. Schematic representation of PTEN protein with exon 5 highlighted in red (bottom). Numbers represent amino acid position. ( B ) Immunoblot for PTEN and phospho-AKT in clones following Pten gRNA transfection. Clones 1.11, 1.12, 1.14 and 1.15 had bi-allelic Pten deletions and showed absent PTEN protein with phosphorylation of AKT at both S473 and T308 following serum starvation. Clones 1.9 and 1.10 had mono-allelic deletions. F3 = ID8 Trp53 −/− . ( C ) Design of guide RNA targeted to exon 2 of Nf1 as previously 29 . Nucleotides in red represent PAM sequence. Schematic representation of Nf1 protein with exon 2 highlighted in red (bottom). Numbers represent amino acid positions. ( D ) Immunoblot for Nf1 in clones following Nf1 gRNA transfection. *; clones 1.20 and 1.23 had bi-allelic indels confirmed by Sanger sequencing, ^; clone 1.6 had single allele deletion. Clones 1.2, 1.3, 1.4, 1.7 and 1.17 were not sequenced. ( E ) Ras-GTP co-immunoprecipitation and phospho-ERK immunoblot in ID8 clones following Nf1 gRNA transfection. *; clones 1.20, 1.23, 1.30 and 1.56 had confirmed bi-allelic indels and demonstrated increased Ras-GTP pulldown and ERK phosphorylation suggestive of activated Ras signalling. ^; clones 1.6 and 1.58 had single allele deletions. ( F ) ID8 Trp53 −/− ; Pten −/− , Trp53 −/− ; Nf1 −/− and Trp53 −/− ; Pten + /− cells were irradiated (10 Gy), fixed and stained for γH2AX and RAD51, and counterstained with DAPI. RAD51 foci were counted in up to 30 untreated and irradiated cells. Bars represent foci per cell (mean +/− SEM); γH2AX (left) and RAD51 (right); dotted lines represents two-fold increase in γH2AX and RAD51 foci/cell relative to untreated cells as above.

Techniques Used: Sequencing, Clone Assay, Transfection, Immunoprecipitation, Irradiation, Staining

26) Product Images from "Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling"

Article Title: Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling

Journal: eLife

doi: 10.7554/eLife.07091

Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and PI3K-Akt signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of Daple-Gαi3 and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007
Figure Legend Snippet: Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and PI3K-Akt signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of Daple-Gαi3 and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007

Techniques Used: Binding Assay, Incubation, Activation Assay, Clone Assay, Expressing, Mutagenesis, Transfection, Lysis, Inhibition

27) Product Images from "Galectin-1 has potential prognostic significance and is implicated in clear cell renal cell carcinoma progression through the HIF/mTOR signaling axis"

Article Title: Galectin-1 has potential prognostic significance and is implicated in clear cell renal cell carcinoma progression through the HIF/mTOR signaling axis

Journal: British Journal of Cancer

doi: 10.1038/bjc.2013.828

A schematic showing a proposed mechanism of Gal-1 involvement in the HIF/mTOR signaling axis in renal cell carcinoma. (1) Increased HIF-1 α because of the hypoxic tumor microenvironment (2) translocates to the nucleus and transcriptionally activates the LGALS1 gene. Gal-1 protein then, by a mechanism that has yet to be elucidated, (3) activates Akt by phosphorylating S473, which in turn phosphorylates mTOR at S2448 and p70S6 kinase to result in increased protein synthesis. This pathway can be blocked by the addition of (4) siRNA that is directed towards Gal-1. We also showed that the pathway can be affected by miRNA as (5) miR-22 can target both Gal-1 and HIF-1 α .
Figure Legend Snippet: A schematic showing a proposed mechanism of Gal-1 involvement in the HIF/mTOR signaling axis in renal cell carcinoma. (1) Increased HIF-1 α because of the hypoxic tumor microenvironment (2) translocates to the nucleus and transcriptionally activates the LGALS1 gene. Gal-1 protein then, by a mechanism that has yet to be elucidated, (3) activates Akt by phosphorylating S473, which in turn phosphorylates mTOR at S2448 and p70S6 kinase to result in increased protein synthesis. This pathway can be blocked by the addition of (4) siRNA that is directed towards Gal-1. We also showed that the pathway can be affected by miRNA as (5) miR-22 can target both Gal-1 and HIF-1 α .

Techniques Used:

28) Product Images from "PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1"

Article Title: PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2011.07.107

PARP1 inhibitors attenuate AKT phosphorylation and its function. (A) Western blot analysis of phospho-AKT S473 and total AKT in U2OS cells in the absence or presence of 15 μM PJ-34 or 10 μM 3-AB for the indicated time. (B) Western blot
Figure Legend Snippet: PARP1 inhibitors attenuate AKT phosphorylation and its function. (A) Western blot analysis of phospho-AKT S473 and total AKT in U2OS cells in the absence or presence of 15 μM PJ-34 or 10 μM 3-AB for the indicated time. (B) Western blot

Techniques Used: Western Blot

29) Product Images from "Akt directly regulates focal adhesion kinase through association and serine phosphorylation: implication for pressure-induced colon cancer metastasis"

Article Title: Akt directly regulates focal adhesion kinase through association and serine phosphorylation: implication for pressure-induced colon cancer metastasis

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00377.2010

Silencing FAK with siRNA prevented the pressure-induced increases in phosphorylation of Akt at S473 ( A ), and FAK-specific inhibitor Y15 prevented pressure-induced increases in Caco-2 cell adhesion ( B ), phosphorylation of FAK at Y397 ( C ), and Akt(S473)
Figure Legend Snippet: Silencing FAK with siRNA prevented the pressure-induced increases in phosphorylation of Akt at S473 ( A ), and FAK-specific inhibitor Y15 prevented pressure-induced increases in Caco-2 cell adhesion ( B ), phosphorylation of FAK at Y397 ( C ), and Akt(S473)

Techniques Used:

30) Product Images from "Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice"

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

Journal: Oncogenesis

doi: 10.1038/oncsis.2012.28

Prostate tissue lacking Abi1 exhibit enhanced proliferation and loss of cellular adhesion markers downstream of WAVE2 complex downregulation. ( a ) Cells and prostate tissues lacking Abi1 exhibit downregulation of WAVE2 complex components but upregulation of Abi2 as indicated by western blotting analysis. Examination of WAVE2 complex integral proteins as indicated in Abi1 KO MEF cells ( a ); and in prostate tissue ( b ). Please note co-incidental downregulation of E-cadherin in prostate tissue and upregulation of Abi2 evident at 12 months in Abi1 KO MEF cells. Anterior prostates were analyzed at 10 and 12 months, ‘−' indicates absence of probasin Cre recombinase transgene and ‘+' indicates its presence in Abi1 floxed mice. ( b ) Enhanced cellular proliferation in prostate tissue lacking Abi1 correlates with enhanced Abi2 levels in prostate tissue. Top: immunostaining of Abi1 KO prostate tissues with Abi2 antibody. Staining with Abi2 antibody (P20, Santa Cruz Biotechnology) reveals striking upregulation of Abi2 levels in Abi1 KO lesions. Middle: enhanced proliferation as indicated by positive staining with Ki67. Bottom: immunostaining with phospho-Akt S473 antibody indicating staining in the PIN lesion (circle). Right panels, prostate tissue from Abi1 lacking (Abi1(fl/fl);PBCre(+)); left panels, prostate tissue from with Abi1 expression (Abi1(fl/fl);PBCre(+)). Staining of tissue was performed as described in Materials and methods.
Figure Legend Snippet: Prostate tissue lacking Abi1 exhibit enhanced proliferation and loss of cellular adhesion markers downstream of WAVE2 complex downregulation. ( a ) Cells and prostate tissues lacking Abi1 exhibit downregulation of WAVE2 complex components but upregulation of Abi2 as indicated by western blotting analysis. Examination of WAVE2 complex integral proteins as indicated in Abi1 KO MEF cells ( a ); and in prostate tissue ( b ). Please note co-incidental downregulation of E-cadherin in prostate tissue and upregulation of Abi2 evident at 12 months in Abi1 KO MEF cells. Anterior prostates were analyzed at 10 and 12 months, ‘−' indicates absence of probasin Cre recombinase transgene and ‘+' indicates its presence in Abi1 floxed mice. ( b ) Enhanced cellular proliferation in prostate tissue lacking Abi1 correlates with enhanced Abi2 levels in prostate tissue. Top: immunostaining of Abi1 KO prostate tissues with Abi2 antibody. Staining with Abi2 antibody (P20, Santa Cruz Biotechnology) reveals striking upregulation of Abi2 levels in Abi1 KO lesions. Middle: enhanced proliferation as indicated by positive staining with Ki67. Bottom: immunostaining with phospho-Akt S473 antibody indicating staining in the PIN lesion (circle). Right panels, prostate tissue from Abi1 lacking (Abi1(fl/fl);PBCre(+)); left panels, prostate tissue from with Abi1 expression (Abi1(fl/fl);PBCre(+)). Staining of tissue was performed as described in Materials and methods.

Techniques Used: Western Blot, Mouse Assay, Immunostaining, Staining, Expressing

Enhanced activation of Akt and colony formation of cells lacking Abi1. ( a , b ) MEF cells lacking Abi1 exhibit enhanced sensitivity to activation of phospho-Akt downstream from EGFR receptor. Cells were starved overnight and subsequently incubated with epidermal growth factor (10 ng/ml) for indicated amounts of time. ( a ) Cell lysates were prepared as described in Materials and methods, and proteins were analyzed by western blotting with indicated antibodies. ( b ) Quantification of phospho-Akt S473 levels indicate significant increase of p-Akt S473 signal of the Abi1 KO clone #3–11 at 1 min and 5 min ( P
Figure Legend Snippet: Enhanced activation of Akt and colony formation of cells lacking Abi1. ( a , b ) MEF cells lacking Abi1 exhibit enhanced sensitivity to activation of phospho-Akt downstream from EGFR receptor. Cells were starved overnight and subsequently incubated with epidermal growth factor (10 ng/ml) for indicated amounts of time. ( a ) Cell lysates were prepared as described in Materials and methods, and proteins were analyzed by western blotting with indicated antibodies. ( b ) Quantification of phospho-Akt S473 levels indicate significant increase of p-Akt S473 signal of the Abi1 KO clone #3–11 at 1 min and 5 min ( P

Techniques Used: Activation Assay, Incubation, Western Blot

31) Product Images from "Developmental vascular regression is regulated by a Wnt/β-catenin, MYC and CDKN1A pathway that controls cell proliferation and cell death"

Article Title: Developmental vascular regression is regulated by a Wnt/β-catenin, MYC and CDKN1A pathway that controls cell proliferation and cell death

Journal: Development (Cambridge, England)

doi: 10.1242/dev.154898

Wnt/β-catenin pathway interaction with CDKN1A and the apoptotic response. (A,B) BMVECs isolated from control ( Lrp5 fl/fl ; Lrp6 fl/fl ) and conditional knockout ( Pdgfb-icreERT2; Lrp5 fl/fl ; Lrp6 fl/fl ) mice labeled for nuclei (blue) and cadherin 5 (CDH5, green). (C-F) BMVECs from the indicated genotypes and shRNA treatments labeled for nuclei (blue) and EdU incorporation (red). (G) Quantification of EdU incorporation in BMVECs from the indicated genotypes and shRNA treatments ( n ≥3). Statistical significance was determined using two-way ANOVA with Bonferroni post-hoc test. Data are mean±s.e.m. (H) Immunoblots from lysates of BMVECs treated with combinations of recombinant VEGFA (rVEGFA) and an shRNA to Cdkn1a (C, control shRNA; Cdk, shRNA to Cdkn1a ). In descending order, the immunoblots show detection of CDKN1A, activated caspase 3, VEGFR2, phospho-VEGFR2, phospho-T308-Akt, phospho-S473-Akt and total Akt.
Figure Legend Snippet: Wnt/β-catenin pathway interaction with CDKN1A and the apoptotic response. (A,B) BMVECs isolated from control ( Lrp5 fl/fl ; Lrp6 fl/fl ) and conditional knockout ( Pdgfb-icreERT2; Lrp5 fl/fl ; Lrp6 fl/fl ) mice labeled for nuclei (blue) and cadherin 5 (CDH5, green). (C-F) BMVECs from the indicated genotypes and shRNA treatments labeled for nuclei (blue) and EdU incorporation (red). (G) Quantification of EdU incorporation in BMVECs from the indicated genotypes and shRNA treatments ( n ≥3). Statistical significance was determined using two-way ANOVA with Bonferroni post-hoc test. Data are mean±s.e.m. (H) Immunoblots from lysates of BMVECs treated with combinations of recombinant VEGFA (rVEGFA) and an shRNA to Cdkn1a (C, control shRNA; Cdk, shRNA to Cdkn1a ). In descending order, the immunoblots show detection of CDKN1A, activated caspase 3, VEGFR2, phospho-VEGFR2, phospho-T308-Akt, phospho-S473-Akt and total Akt.

Techniques Used: Isolation, Knock-Out, Mouse Assay, Labeling, shRNA, Western Blot, Recombinant

32) Product Images from "Aberrant REDD1-mTORC1 responses to insulin in skeletal muscle from Type 2 diabetics"

Article Title: Aberrant REDD1-mTORC1 responses to insulin in skeletal muscle from Type 2 diabetics

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

doi: 10.1152/ajpregu.00285.2015

Limited insulin activation of Akt in Type 2 diabetes (T2D) human skeletal muscle. A vastus lateralis skeletal muscle biopsy was obtained from lean, control and obese, or T2D subjects under basal and after a 2-h hyperinsulinemic (40 mU·m −2 ·min −1 )-euglycemic (5 mM) clamp, then frozen in liquid nitrogen. A total homogenate of this muscle biopsy was analyzed by Western blot analysis for phospho-Akt S473 and normalized to GADPH relative to control and percent change (Δ denotes percent change or delta) of basal-to-insulin phospho-Akt S473 for the respective group. Representative blots are shown. * P
Figure Legend Snippet: Limited insulin activation of Akt in Type 2 diabetes (T2D) human skeletal muscle. A vastus lateralis skeletal muscle biopsy was obtained from lean, control and obese, or T2D subjects under basal and after a 2-h hyperinsulinemic (40 mU·m −2 ·min −1 )-euglycemic (5 mM) clamp, then frozen in liquid nitrogen. A total homogenate of this muscle biopsy was analyzed by Western blot analysis for phospho-Akt S473 and normalized to GADPH relative to control and percent change (Δ denotes percent change or delta) of basal-to-insulin phospho-Akt S473 for the respective group. Representative blots are shown. * P

Techniques Used: Activation Assay, Western Blot

33) Product Images from "Decoding the functional Fes kinase signaling network topology in a lymphocyte model"

Article Title: Decoding the functional Fes kinase signaling network topology in a lymphocyte model

Journal: bioRxiv

doi: 10.1101/125088

(A) Western blot analysis of endogenous Fes kinase from RAW684.3 (macrophage) and DG75 (B-lymphocyte) cell lines (Arrow indicates Fes protein band). Quantification of cellular protein levels is normalized to Gapdh and shown in the lower panel. (B) Western blot validation of activating phosphorylation sites on known BCR responders such as Syk, Btk, Lyn and CD19 showed a strong engagement of diverse signaling cascades following BCR stimulation of DG75 cells. (C) Downstream signaling pathways such as the recruitment of Akt to the membrane (Dok1 localization to the membrane is shown as a control for membrane preparations) and (D) activation of Akt and Erk pathways, as measured by phosphorylation of S473 and T202/Y204 respectively, confirmed DG75’s suitability as a model system for BCR activation and signal processing.
Figure Legend Snippet: (A) Western blot analysis of endogenous Fes kinase from RAW684.3 (macrophage) and DG75 (B-lymphocyte) cell lines (Arrow indicates Fes protein band). Quantification of cellular protein levels is normalized to Gapdh and shown in the lower panel. (B) Western blot validation of activating phosphorylation sites on known BCR responders such as Syk, Btk, Lyn and CD19 showed a strong engagement of diverse signaling cascades following BCR stimulation of DG75 cells. (C) Downstream signaling pathways such as the recruitment of Akt to the membrane (Dok1 localization to the membrane is shown as a control for membrane preparations) and (D) activation of Akt and Erk pathways, as measured by phosphorylation of S473 and T202/Y204 respectively, confirmed DG75’s suitability as a model system for BCR activation and signal processing.

Techniques Used: Western Blot, Activation Assay

34) Product Images from "A TLR/AKT/FoxO3 immune tolerance–like pathway disrupts the repair capacity of oligodendrocyte progenitors"

Article Title: A TLR/AKT/FoxO3 immune tolerance–like pathway disrupts the repair capacity of oligodendrocyte progenitors

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI94158

bHAf regulates AKT in a tolerance-like manner. ( A–C ) bHAf induces persistent AKT dephosphorylation in rat slices treated with bHAf (100 nM). Representative blots and quantification after probing with pAKT-S473, pAKT-T308, pERK1/2, AKT (total AKT), and actin antibodies. Note the transient increase in AKT phosphorylation followed by persistent reduction to control levels at 4 hours ( A ), 24 hours ( B ), and 5 or 9 days ( C ). ( D ) BDNF (50 ng/ml) induces persistent AKT phosphorylation at 30 minutes, and 4 or 24 hours. ( E ) Experimental design to determine whether BDNF can overcome bHAf-mediated AKT desensitization (left); and representative blots (right). ( F ) Chronic WMI leads to persistent AKT dephosphorylation that normalizes with delayed partial myelination. Representative blots and quantification from 3 rats that underwent H-I at P3 versus control (C), comparing AKT phosphorylation in lesion hemisphere at P4, P7, P10, P14, and P21. Actin was the loading control. ( G ) Progressive recovery of myelination following WMI. Representative images depicting MBP staining at P10, P14, and P21 (asterisks indicate H-I hemisphere). A – E : n = 3 independent studies from 3 separate litters; 2 slices/condition. F : n = 3 animals (control) and n = 6 animals (H-I). G : n = 9 animals/condition. * P
Figure Legend Snippet: bHAf regulates AKT in a tolerance-like manner. ( A–C ) bHAf induces persistent AKT dephosphorylation in rat slices treated with bHAf (100 nM). Representative blots and quantification after probing with pAKT-S473, pAKT-T308, pERK1/2, AKT (total AKT), and actin antibodies. Note the transient increase in AKT phosphorylation followed by persistent reduction to control levels at 4 hours ( A ), 24 hours ( B ), and 5 or 9 days ( C ). ( D ) BDNF (50 ng/ml) induces persistent AKT phosphorylation at 30 minutes, and 4 or 24 hours. ( E ) Experimental design to determine whether BDNF can overcome bHAf-mediated AKT desensitization (left); and representative blots (right). ( F ) Chronic WMI leads to persistent AKT dephosphorylation that normalizes with delayed partial myelination. Representative blots and quantification from 3 rats that underwent H-I at P3 versus control (C), comparing AKT phosphorylation in lesion hemisphere at P4, P7, P10, P14, and P21. Actin was the loading control. ( G ) Progressive recovery of myelination following WMI. Representative images depicting MBP staining at P10, P14, and P21 (asterisks indicate H-I hemisphere). A – E : n = 3 independent studies from 3 separate litters; 2 slices/condition. F : n = 3 animals (control) and n = 6 animals (H-I). G : n = 9 animals/condition. * P

Techniques Used: De-Phosphorylation Assay, Staining

35) Product Images from "A Kinome-Wide RNAi Screen in Drosophila Glia Reveals That the RIO Kinases Mediate Cell Proliferation and Survival through TORC2-Akt Signaling in Glioblastoma"

Article Title: A Kinome-Wide RNAi Screen in Drosophila Glia Reveals That the RIO Kinases Mediate Cell Proliferation and Survival through TORC2-Akt Signaling in Glioblastoma

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003253

RIOK2 overexpression in GBM tumors is associated with Akt signaling. (A–E) Immunohistochemistry for RIOK2 (reddish brown) showing cytoplasmic and submembraneous enrichment for RIOK2 in tumor cells. Hematoxilin counterstain, (A) GBM39 tissue, from a subcutanteous xenograft, showing RIOK2 staining in tumor cells (arrows), which formed lobules delineated by RIOK2-negative host stromal cells. (B) ΔEGFR-positive human GBM with RIOK2-positive giant cell component (inset shows a conspicuous giant cell), and RIOK2-negative tumor stroma composed of abnormal blood vessels (“BV”). (C) ΔEGFR-positive human GBM, abnormal mitotic cells with high RIOK2 staining denoted with asterisks and shown in inset close-up. (D) ΔEGFR-positive human GBM, lower magnification to highlight enriched RIOK2 in pseudopallisades (“PSS”), inset shows enriched RIOK2 staining present in dense cellular regions of pseudopallisades. (E) RIOK2 expression in an EGFR-overexpressing human GBM with (F) matched normal control tissue from the same surgical specimen, arrows denote normal astrocytes (recognized by their open nuclei). (G) RIOK2 expression in an EGFR-negative/Akt-S473-P-positive GBM shown alongside (H) another example of normal control brain tissue. Arrows denote normal neuronal cells (recognized by their basophilic cell bodies) with low/undetectable RIOK2 expression. (I) and (J) examples of Akt-S473-P and EGFR-Y1068-P immunoreactivity in RIOK2-positive GBM tumor specimens. (K) a RIOK2- negative GBM with a negative abnormal blood vessel (“BV”). (L) Statistical analysis of RIOK2-positive and negative tumor specimens showing a significant correlation between RIOK2 expression and phosphorylation of EGFR at Tyrosine-1068 and phosphorylation of Akt at Serine-473. More stains from tumors shown in Figures S10 and S11.
Figure Legend Snippet: RIOK2 overexpression in GBM tumors is associated with Akt signaling. (A–E) Immunohistochemistry for RIOK2 (reddish brown) showing cytoplasmic and submembraneous enrichment for RIOK2 in tumor cells. Hematoxilin counterstain, (A) GBM39 tissue, from a subcutanteous xenograft, showing RIOK2 staining in tumor cells (arrows), which formed lobules delineated by RIOK2-negative host stromal cells. (B) ΔEGFR-positive human GBM with RIOK2-positive giant cell component (inset shows a conspicuous giant cell), and RIOK2-negative tumor stroma composed of abnormal blood vessels (“BV”). (C) ΔEGFR-positive human GBM, abnormal mitotic cells with high RIOK2 staining denoted with asterisks and shown in inset close-up. (D) ΔEGFR-positive human GBM, lower magnification to highlight enriched RIOK2 in pseudopallisades (“PSS”), inset shows enriched RIOK2 staining present in dense cellular regions of pseudopallisades. (E) RIOK2 expression in an EGFR-overexpressing human GBM with (F) matched normal control tissue from the same surgical specimen, arrows denote normal astrocytes (recognized by their open nuclei). (G) RIOK2 expression in an EGFR-negative/Akt-S473-P-positive GBM shown alongside (H) another example of normal control brain tissue. Arrows denote normal neuronal cells (recognized by their basophilic cell bodies) with low/undetectable RIOK2 expression. (I) and (J) examples of Akt-S473-P and EGFR-Y1068-P immunoreactivity in RIOK2-positive GBM tumor specimens. (K) a RIOK2- negative GBM with a negative abnormal blood vessel (“BV”). (L) Statistical analysis of RIOK2-positive and negative tumor specimens showing a significant correlation between RIOK2 expression and phosphorylation of EGFR at Tyrosine-1068 and phosphorylation of Akt at Serine-473. More stains from tumors shown in Figures S10 and S11.

Techniques Used: Over Expression, Immunohistochemistry, Staining, Expressing

36) Product Images from "LAMB3 mediates apoptotic, proliferative, invasive, and metastatic behaviors in pancreatic cancer by regulating the PI3K/Akt signaling pathway"

Article Title: LAMB3 mediates apoptotic, proliferative, invasive, and metastatic behaviors in pancreatic cancer by regulating the PI3K/Akt signaling pathway

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1320-z

LAMB3 regulates PI3K-mediated Akt phosphorylation and epithelial‒mesenchymal transition (EMT) in pancreatic cells. a The levels of total Akt, p-Akt-S473, and the EMT-related proteins N-cadherin, E-cadherin, Snail, Slug, and vimentin were examined by western blot analysis, as were those of the b tumor invasion and migration-related proteins MMP2 and MMP9. c Invasion and migration were examined by transwell assays in control and LAMB3-overexpressing PANC-1 and MIA PaCa-2 cells after LY294002 (10 μM) treatment for 48 h. d EMT-related proteins and the PI3K/Akt-related proteins PI3K, PDK2, total Akt, and p-Akt-S473 were evaluated by western blot analysis in the LAMB3D, LAMB3U, and NC groups. e After treatment with or without 10 μM LY294002 for 48 h, the expression of EMT-related proteins, including N-cadherin, E-cadherin, Snail, Slug, Akt, and p-Akt-S473, was evaluated by western blot analysis in the LAMB3U and NC groups. Each figure is representative of three independent experiments
Figure Legend Snippet: LAMB3 regulates PI3K-mediated Akt phosphorylation and epithelial‒mesenchymal transition (EMT) in pancreatic cells. a The levels of total Akt, p-Akt-S473, and the EMT-related proteins N-cadherin, E-cadherin, Snail, Slug, and vimentin were examined by western blot analysis, as were those of the b tumor invasion and migration-related proteins MMP2 and MMP9. c Invasion and migration were examined by transwell assays in control and LAMB3-overexpressing PANC-1 and MIA PaCa-2 cells after LY294002 (10 μM) treatment for 48 h. d EMT-related proteins and the PI3K/Akt-related proteins PI3K, PDK2, total Akt, and p-Akt-S473 were evaluated by western blot analysis in the LAMB3D, LAMB3U, and NC groups. e After treatment with or without 10 μM LY294002 for 48 h, the expression of EMT-related proteins, including N-cadherin, E-cadherin, Snail, Slug, Akt, and p-Akt-S473, was evaluated by western blot analysis in the LAMB3U and NC groups. Each figure is representative of three independent experiments

Techniques Used: Western Blot, Migration, Expressing

37) Product Images from "G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth"

Article Title: G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth

Journal: Blood

doi: 10.1182/blood-2005-04-1612

Time course for G-CSF-induced activation of Lyn, Akt, and ERK1/2. Ba/F3GR cells were stimulated with or without 100 ng/mL G-CSF for indicated time periods, then whole-cell lysates were prepared. (A) Lyn activation. Immunoblotting (IB) was performed using anti-phospho-Src Y416 antibody, which detects the activated state of Lyn, and anti-phospho-Lyn Y507, which detects the nonactivated state of Lyn. The blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes. (B) Akt activation. Immunoblotting was performed using anti-phospho-Akt S473 antibody and reprobed with anti-Akt antibody. (C) ERK1/2 activation. Immunoblotting was performed using anti-phospho-ERK1/2 2T202/Y204 antibody and reprobed with antiactin antibody. Comparable results were observed in 4 independent experiments.
Figure Legend Snippet: Time course for G-CSF-induced activation of Lyn, Akt, and ERK1/2. Ba/F3GR cells were stimulated with or without 100 ng/mL G-CSF for indicated time periods, then whole-cell lysates were prepared. (A) Lyn activation. Immunoblotting (IB) was performed using anti-phospho-Src Y416 antibody, which detects the activated state of Lyn, and anti-phospho-Lyn Y507, which detects the nonactivated state of Lyn. The blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes. (B) Akt activation. Immunoblotting was performed using anti-phospho-Akt S473 antibody and reprobed with anti-Akt antibody. (C) ERK1/2 activation. Immunoblotting was performed using anti-phospho-ERK1/2 2T202/Y204 antibody and reprobed with antiactin antibody. Comparable results were observed in 4 independent experiments.

Techniques Used: Activation Assay

Effect of kinase inhibitors on G-CSF-induced activation of Akt or ERK1/2. (A) Inhibition of downstream substrates of Akt, mTOR, and GSK3 by a specific Akt inhibitor A838450. Ba/F3GR cells were pretreated for 1 hour with the indicated concentrations of Akt inhibitor A838450 or DMSO (the diluent control), then stimulated with or without 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-mTOR S2448 or anti-phospho-GSK3 S21/9 antibodies. Blots were stripped and reprobed with anti-mTOR or anti-GSK3 antibody to demonstrate comparable protein loading. (B) Akt inhibition. Ba/F3GR cells were pretreated for 1 hour with the indicated concentrations of Akt inhibitor A838450 or DMSO (the diluent control), then left unstimulated or stimulated with 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-AktS473 and anti-phospho-ERK1/2 T202/Y204 antibody. Blot was stripped and reprobed with anti-Akt or antiactin antibody to demonstrate comparable protein loading. (C) ERK1/2 inhibition. Ba/F3GR cells were pretreated with the indicated concentrations of MEK inhibitor or DMSO (diluent control) for 1 hour and then stimulated with 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-ERK1/2 T202/Y204 antibody or anti-phospho-Akt S473. Blot was stripped and reprobed with antiactin antibody to demonstrate comparable protein loading.
Figure Legend Snippet: Effect of kinase inhibitors on G-CSF-induced activation of Akt or ERK1/2. (A) Inhibition of downstream substrates of Akt, mTOR, and GSK3 by a specific Akt inhibitor A838450. Ba/F3GR cells were pretreated for 1 hour with the indicated concentrations of Akt inhibitor A838450 or DMSO (the diluent control), then stimulated with or without 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-mTOR S2448 or anti-phospho-GSK3 S21/9 antibodies. Blots were stripped and reprobed with anti-mTOR or anti-GSK3 antibody to demonstrate comparable protein loading. (B) Akt inhibition. Ba/F3GR cells were pretreated for 1 hour with the indicated concentrations of Akt inhibitor A838450 or DMSO (the diluent control), then left unstimulated or stimulated with 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-AktS473 and anti-phospho-ERK1/2 T202/Y204 antibody. Blot was stripped and reprobed with anti-Akt or antiactin antibody to demonstrate comparable protein loading. (C) ERK1/2 inhibition. Ba/F3GR cells were pretreated with the indicated concentrations of MEK inhibitor or DMSO (diluent control) for 1 hour and then stimulated with 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-ERK1/2 T202/Y204 antibody or anti-phospho-Akt S473. Blot was stripped and reprobed with antiactin antibody to demonstrate comparable protein loading.

Techniques Used: Activation Assay, Inhibition

Enhanced ROS production in cells expressing a truncated G-CSF receptor. (A) Increased ROS production in cells expressing the truncated G-CSF receptor. Stable transfectants of Ba/F3 cells, expressing comparable levels of either wild-type or truncated G-CSF receptor, were labeled with dihydrorhodamine123 and unstimulated or stimulated with 100 ng/mL G-CSF for indicated periods of time. The relative ROS level induced by G-CSF was determined by monitoring the increased fluorescence (means ± SD) of rhodamine123 in the cells. (B) Truncated G-CSF receptor knock-in mice showed enhanced ROS production. Neutrophils from bone marrow of wild-type and GRΔ715 mice were loaded with DHR followed by stimulation with 100 ng/mL G-CSF for 60 minutes (green) or not (purple). One representative is shown in the left panel; quantitative results (means ± SD of 3 independent experiments) are shown in the right panel. (C) G-CSF-induced sustained activation of Lyn in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-Src Y416 antibody, which detects the activated state of Lyn. Blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes. (D) G-CSF-induced activation of Akt in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-Akt S473 antibody, then the blot was stripped and reprobed with anti-Akt antibody to demonstrate comparable levels of protein loaded in respective lanes. (E) G-CSF-induced activation of ERK1/2 in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-ERK1/2 T202T204 antibody, then the blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes.
Figure Legend Snippet: Enhanced ROS production in cells expressing a truncated G-CSF receptor. (A) Increased ROS production in cells expressing the truncated G-CSF receptor. Stable transfectants of Ba/F3 cells, expressing comparable levels of either wild-type or truncated G-CSF receptor, were labeled with dihydrorhodamine123 and unstimulated or stimulated with 100 ng/mL G-CSF for indicated periods of time. The relative ROS level induced by G-CSF was determined by monitoring the increased fluorescence (means ± SD) of rhodamine123 in the cells. (B) Truncated G-CSF receptor knock-in mice showed enhanced ROS production. Neutrophils from bone marrow of wild-type and GRΔ715 mice were loaded with DHR followed by stimulation with 100 ng/mL G-CSF for 60 minutes (green) or not (purple). One representative is shown in the left panel; quantitative results (means ± SD of 3 independent experiments) are shown in the right panel. (C) G-CSF-induced sustained activation of Lyn in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-Src Y416 antibody, which detects the activated state of Lyn. Blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes. (D) G-CSF-induced activation of Akt in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-Akt S473 antibody, then the blot was stripped and reprobed with anti-Akt antibody to demonstrate comparable levels of protein loaded in respective lanes. (E) G-CSF-induced activation of ERK1/2 in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-ERK1/2 T202T204 antibody, then the blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes.

Techniques Used: Expressing, Labeling, Fluorescence, Knock-In, Mouse Assay, Activation Assay

38) Product Images from "Protein Kinase C alpha (PKC?) dependent signaling mediates endometrial cancer cell growth and tumorigenesis"

Article Title: Protein Kinase C alpha (PKC?) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

Journal: International journal of cancer. Journal international du cancer

doi: 10.1002/ijc.24633

Effects of Akt and ERK on p21 and p27 expression. Ishikawa cells were serum starved for 8h before treatment with vehicle (DMSO); Akt inhibitor Akti-1/2 (5μM); or the MEK1/MEK2 inhibitor U0126 (5 nM) to block ERK activation. Cells were treated in medium containing 0.5% serum and vehicle or inhibitors were added fresh at 24 and 48h time points. Treated cells were harvested at 24, 48 and 72h and probed using antibodies to CDK inhibitors p21 and p27; phospho-ERK (P-ERK), Akt phosphorylated on S473 (P-Akt (S473)), total Akt and total ERK. β-actin is loading control. Blots shown are representative of two separate experiments.
Figure Legend Snippet: Effects of Akt and ERK on p21 and p27 expression. Ishikawa cells were serum starved for 8h before treatment with vehicle (DMSO); Akt inhibitor Akti-1/2 (5μM); or the MEK1/MEK2 inhibitor U0126 (5 nM) to block ERK activation. Cells were treated in medium containing 0.5% serum and vehicle or inhibitors were added fresh at 24 and 48h time points. Treated cells were harvested at 24, 48 and 72h and probed using antibodies to CDK inhibitors p21 and p27; phospho-ERK (P-ERK), Akt phosphorylated on S473 (P-Akt (S473)), total Akt and total ERK. β-actin is loading control. Blots shown are representative of two separate experiments.

Techniques Used: Expressing, Blocking Assay, Activation Assay

PKCα knockdown inhibits Akt and ERK activation. (a) Ishikawa clones expressing shRNA constructs directed against luciferase (Luc) or PKCα were deprived of serum for 24h before cell harvest. Western blots were probed with phospho-specific antibodies to residues S473 of Akt (P-Akt (S473)) and S9 of GSK-3β (P-GSK-3β (S9)), and antibodies to total Akt and total GSK-3β as indicated. (b) Quantitation of P-AKT/total AKT, results are mean ± s.d. N=6,**p
Figure Legend Snippet: PKCα knockdown inhibits Akt and ERK activation. (a) Ishikawa clones expressing shRNA constructs directed against luciferase (Luc) or PKCα were deprived of serum for 24h before cell harvest. Western blots were probed with phospho-specific antibodies to residues S473 of Akt (P-Akt (S473)) and S9 of GSK-3β (P-GSK-3β (S9)), and antibodies to total Akt and total GSK-3β as indicated. (b) Quantitation of P-AKT/total AKT, results are mean ± s.d. N=6,**p

Techniques Used: Activation Assay, Clone Assay, Expressing, shRNA, Construct, Luciferase, Western Blot, Quantitation Assay

39) Product Images from "Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss"

Article Title: Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss

Journal: eLife

doi: 10.7554/eLife.17635

Membrane targeting p110α vectors selectively enrich p110α in the desired microdomain. ( A ) DKO+p110α-Lyn and DKO+p110α-Ras MEFs were lysed and fractionated into triton sensitive and triton resistant membrane fractions. WCLs were analyzed to display overall levels of protein expression. Soluble, triton soluble (membrane) and resistant membrane fractions (DRM) were analyzed in immunoblots; anti-Rac1 and anti-Caveolin1 antibodies were used as markers for detergent resistant membrane (DRM) rafts, whereas anti-TfnR immunoblot depicts enrichment of nonraft membranes. Anti-tubulin immunoblot serves as a marker for soluble fractions. ( B ) The indicated DKO add-back MEFs were seeded on coverslips, starved and stimulated with LPA; membrane associated Akt phosphorylation at T308 and S473 was detected using anti-p-Akt T308 and p-Akt S473 antibodies (green). Anti-HA antibodies (red) depicted expression levels of the add-back vectors. DNA is shown in blue. Scale bar; 20 µm. On the right, anti-p-Akt T308 and S473 signals on the cell membrane was quantified upon LPA stimulation and the relative corrected total membrane fluorescence was depicted. Results denote mean of 3 independent experiments with standard deviation. ** p
Figure Legend Snippet: Membrane targeting p110α vectors selectively enrich p110α in the desired microdomain. ( A ) DKO+p110α-Lyn and DKO+p110α-Ras MEFs were lysed and fractionated into triton sensitive and triton resistant membrane fractions. WCLs were analyzed to display overall levels of protein expression. Soluble, triton soluble (membrane) and resistant membrane fractions (DRM) were analyzed in immunoblots; anti-Rac1 and anti-Caveolin1 antibodies were used as markers for detergent resistant membrane (DRM) rafts, whereas anti-TfnR immunoblot depicts enrichment of nonraft membranes. Anti-tubulin immunoblot serves as a marker for soluble fractions. ( B ) The indicated DKO add-back MEFs were seeded on coverslips, starved and stimulated with LPA; membrane associated Akt phosphorylation at T308 and S473 was detected using anti-p-Akt T308 and p-Akt S473 antibodies (green). Anti-HA antibodies (red) depicted expression levels of the add-back vectors. DNA is shown in blue. Scale bar; 20 µm. On the right, anti-p-Akt T308 and S473 signals on the cell membrane was quantified upon LPA stimulation and the relative corrected total membrane fluorescence was depicted. Results denote mean of 3 independent experiments with standard deviation. ** p

Techniques Used: Expressing, Western Blot, Marker, Fluorescence, Standard Deviation

Raft targeting of Rac1-binding deficient p110β rescues Akt activation in GPCR signaling. ( A ) Schematic demonstration of p110β-Lyn domain membrane targeting vectors. ( B ) Lysates from the indicated MEFs were processed and analyzed for expression of p110α and β. On the right, DKO MEFs expressing the indicated p110β alleles were fractionated into soluble, triton sensitive and triton resistant fractions. Triton resistant fractions were analyzed in immunoblots; anti-HA antibodies were used to visualize the abundance of the p110β variants in those fractions. Anti-Rac1 antibody was used to demonstrate raft enrichment, whereas anti-TfnR immunoblot depicts contamination with nonraft membranes. Anti-actin immunoblot serves as loading control. ( C ) The indicated add-back MEFs were starved and stimulated with serum or LPA. Anti-p-Akt immunoblots on T308 and S473 display level of Akt activation and anti-p-Erk1/2 antibodies (for T202/Y204) depicts activation of MAPK pathway. On the right, density ratios of the normalized fold-increase in baseline Akt phosphorylation at T308 and S473 in starved vs. LPA stimulated states were quantified (mean of 3 independent experiments with standard deviation). ** p
Figure Legend Snippet: Raft targeting of Rac1-binding deficient p110β rescues Akt activation in GPCR signaling. ( A ) Schematic demonstration of p110β-Lyn domain membrane targeting vectors. ( B ) Lysates from the indicated MEFs were processed and analyzed for expression of p110α and β. On the right, DKO MEFs expressing the indicated p110β alleles were fractionated into soluble, triton sensitive and triton resistant fractions. Triton resistant fractions were analyzed in immunoblots; anti-HA antibodies were used to visualize the abundance of the p110β variants in those fractions. Anti-Rac1 antibody was used to demonstrate raft enrichment, whereas anti-TfnR immunoblot depicts contamination with nonraft membranes. Anti-actin immunoblot serves as loading control. ( C ) The indicated add-back MEFs were starved and stimulated with serum or LPA. Anti-p-Akt immunoblots on T308 and S473 display level of Akt activation and anti-p-Erk1/2 antibodies (for T202/Y204) depicts activation of MAPK pathway. On the right, density ratios of the normalized fold-increase in baseline Akt phosphorylation at T308 and S473 in starved vs. LPA stimulated states were quantified (mean of 3 independent experiments with standard deviation). ** p

Techniques Used: Binding Assay, Activation Assay, Expressing, Western Blot, Standard Deviation

Raft-excluded p110β fails to induce activatory Akt phosphorylation upon GPCR stimulation. Indicated DKO add-back MEFs were seeded on coverslips, starved and stimulated with LPA; membrane associated Akt phosphorylation at T308 and S473 was detected using anti-p-Akt T308 and p-Akt S473 antibodies (green). Anti-HA antibodies (red) depicted expression levels of the add-back vectors. DNA is shown in blue. Scale bar; 20 µm. On the right, anti-p-Akt T308 and S473 signals on the cell membrane was quantified upon LPA stimulation and the relative corrected total membrane fluorescence was depicted. Results denote mean of 3 independent experiments with standard deviation. * p
Figure Legend Snippet: Raft-excluded p110β fails to induce activatory Akt phosphorylation upon GPCR stimulation. Indicated DKO add-back MEFs were seeded on coverslips, starved and stimulated with LPA; membrane associated Akt phosphorylation at T308 and S473 was detected using anti-p-Akt T308 and p-Akt S473 antibodies (green). Anti-HA antibodies (red) depicted expression levels of the add-back vectors. DNA is shown in blue. Scale bar; 20 µm. On the right, anti-p-Akt T308 and S473 signals on the cell membrane was quantified upon LPA stimulation and the relative corrected total membrane fluorescence was depicted. Results denote mean of 3 independent experiments with standard deviation. * p

Techniques Used: Expressing, Fluorescence, Standard Deviation

40) Product Images from "Acute Hyperglycemia Abolishes Ischemic Preconditioning by Inhibiting Akt Phosphorylation: Normalizing Blood Glucose before Ischemia Restores Ischemic Preconditioning"

Article Title: Acute Hyperglycemia Abolishes Ischemic Preconditioning by Inhibiting Akt Phosphorylation: Normalizing Blood Glucose before Ischemia Restores Ischemic Preconditioning

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2013/329183

Myocardial phospho-Akt S473 to pan-Akt ratios in the indicated experimental groups. The ratio of phospho-Akt S473 to pan-Akt (the bar graph) was measured by densitometry, where the pan-AKT inputs were normalized to 1.
Figure Legend Snippet: Myocardial phospho-Akt S473 to pan-Akt ratios in the indicated experimental groups. The ratio of phospho-Akt S473 to pan-Akt (the bar graph) was measured by densitometry, where the pan-AKT inputs were normalized to 1.

Techniques Used:

Myocardial phospho-Akt S473 to pan-Akt ratios in mice treated with CCPA.
Figure Legend Snippet: Myocardial phospho-Akt S473 to pan-Akt ratios in mice treated with CCPA.

Techniques Used: Mouse Assay

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    Cell Signaling Technology Inc rabbit anti human pan 4ebp1
    PRE, W3, and W6 mTORc1 signaling marker differences between LOW and HIGH responders. (A–D) Phosphorylated (p-) mTORc1 targets. There were no significant cluster effects or cluster × time interactions for said markers. p-mTOR and <t>p-4EBP1</t> demonstrated significant time effects. (E–H) Total (pan) mTOR, p70s6k, 4EBP1, and AMPKα. Again, there were no significant cluster effects or cluster × time interactions for said markers, although there were significant time effects for pan mTOR, pan p70s6k, and pan AMPKα. Values presented in line graphs are mean (standard deviation) values. (I) Representative Western blot images for a low and high responder.
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    Cell Signaling Technology Inc pan cadherin
    HCV induces OPN and signaling cascade in primary human hepatocytes (PHH). (A, B) Equal amounts of cellular lysates from PHH (lane 1) and HCV-infected PHH (lane 2) were subjected to western blot analysis using anti-OPN, anti-CD44, anti-β3, <t>anti-E-cadherin,</t> anti-N-cadherin, anti-FAK, and anti-pAkt (Ser 473 ). HCV NS3 represents HCV infection and actin was used as protein loading control. (C) Immunofluorescence microscopy of HCV-infected PHH. HCV-infected PHH was incubated with HCV NS5A antibody for 1 h at RT followed by 1 h incubation with secondary HCV NS5A antibody (anti-rabbit Alexa Fluor 546) as describe in Materials and Methods. The magenta color indicates expression of HCV NS5A around the nucleus. Arrow indicates few uninfected cells. DAPI was used as a nuclear stain in blue color. Scale bar 10 µM. The results shown are the representative of three independent experiments.
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    Cell Signaling Technology Inc pan syngap
    Development of isogenic SYNGAP1 knockout hiPSCs. (A ) Cartoon showing clone-specific mutations in the SYNGAP1 gene. (B) Sanger sequencing for one WT clone and two SYNGAP1 mutant clones derived from the CRISPR experiment. ( C ) Whole genome view of iNeurons from WT#6, WT#30, KO#4 and KO#38 clones depicting a copy number value of 2 cross all chromosomes (expect for the Y-chromosome which is not detected) revealing normal (female) karyotype with no chromosomal aberrations. The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number and aberrations (if any). ( D ) Western blots demonstrating <t>SynGAP</t> protein expression from iNeuron or iPSC homogenate. Total refers to signal from an antibody that detects all splice variants and <t>α2</t> refers to signal from an antibody that detects only a specific C-terminal splice variant. ( E ) Quantification of relative intensity of bands normalized to total protein signal. One-way ANOVA with a Kruskal-Wallis test multiple comparisons test H(3)=12.29, p=0.0001; WT#6 vs KO#4: p=0.1876; WT#6 vs WT#30: p > 0.9999; WT#6 vs KO#38: p=0.0140; KO#4 vs WT#30: p=0.5258; KO#4 vs KO#38: p > 0.9999; WT#30 vs KO#38: p=0.0561. n=4 per group. In box-and-whisker plot, the center, boxes and whiskers represent the median, interquartile range, and min to max, respectively. (F) Indels from each clone identified from whole exome sequencing analysis. Indels were identified by clonal sequence differences from the original Cas9 hiPSCs (reference sequence). Indel threshold was determined by at least 50% of the reads differing from the reference sequence with a minimum of at least ten reads. Indels w/ frequency above 0.8 were used to determine frequency of homozygous varaints. ( G ) Normalized mapped reads from the entire coding sequence of the SYNGAP1 gene in the four clones hiPSCs. Red arrow denotes predicted Cas9 cut site. Numbers reflect clonal reads relative to Cas9 hiPSC reads. ( H ) Normalized mapped reads for the same samples around the Cas9 target sequence. ( I ) Genomic PCR to amplify DNA sequence flanking the Cas9 target site.
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    Cell Signaling Technology Inc rabbit anti akt
    Loss of FlnB induces Cdk1 activity changes through β1 <t>integrin-Pi3k/Akt</t> pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p
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    PRE, W3, and W6 mTORc1 signaling marker differences between LOW and HIGH responders. (A–D) Phosphorylated (p-) mTORc1 targets. There were no significant cluster effects or cluster × time interactions for said markers. p-mTOR and p-4EBP1 demonstrated significant time effects. (E–H) Total (pan) mTOR, p70s6k, 4EBP1, and AMPKα. Again, there were no significant cluster effects or cluster × time interactions for said markers, although there were significant time effects for pan mTOR, pan p70s6k, and pan AMPKα. Values presented in line graphs are mean (standard deviation) values. (I) Representative Western blot images for a low and high responder.

    Journal: Frontiers in Physiology

    Article Title: Pre-training Skeletal Muscle Fiber Size and Predominant Fiber Type Best Predict Hypertrophic Responses to 6 Weeks of Resistance Training in Previously Trained Young Men

    doi: 10.3389/fphys.2019.00297

    Figure Lengend Snippet: PRE, W3, and W6 mTORc1 signaling marker differences between LOW and HIGH responders. (A–D) Phosphorylated (p-) mTORc1 targets. There were no significant cluster effects or cluster × time interactions for said markers. p-mTOR and p-4EBP1 demonstrated significant time effects. (E–H) Total (pan) mTOR, p70s6k, 4EBP1, and AMPKα. Again, there were no significant cluster effects or cluster × time interactions for said markers, although there were significant time effects for pan mTOR, pan p70s6k, and pan AMPKα. Values presented in line graphs are mean (standard deviation) values. (I) Representative Western blot images for a low and high responder.

    Article Snippet: Rabbit anti-human phospho-p70s6k (Thr389) (1:1,000; catalog #: 9234; Cell Signaling), rabbit anti-human pan p70s6k (1:1,000; catalog #: 2708; Cell Signaling), rabbit anti-human phospho-4EBP1 (Thr37/46) (1:1,000; catalog #: 2855; Cell Signaling), rabbit anti-human pan 4EBP1 (1:1,000; catalog #: 9644; Cell Signaling), rabbit anti-human phospho-mTOR (Ser2448) (1:1,000; catalog #: 2971; Cell Signaling), rabbit anti-human pan mTOR (1:1,000; catalog #: 2972; Cell Signaling), rabbit anti-human phospho-AMPKα (Thr172) (1:1,000; catalog #: 2535; Cell Signaling), rabbit anti-human pan AMPKα (1:1,000; catalog #: 2532; Cell Signaling), rabbit anti-human androgen receptor (1:1,000; catalog #: 5153; Cell Signaling) and rabbit anti-human ubiquitin (1:1,000; catalog #: 3933; Cell Signaling) were incubated with membranes overnight at 4°C in TBST with 5% bovine serum albumin (BSA).

    Techniques: Marker, Standard Deviation, Western Blot

    HCV induces OPN and signaling cascade in primary human hepatocytes (PHH). (A, B) Equal amounts of cellular lysates from PHH (lane 1) and HCV-infected PHH (lane 2) were subjected to western blot analysis using anti-OPN, anti-CD44, anti-β3, anti-E-cadherin, anti-N-cadherin, anti-FAK, and anti-pAkt (Ser 473 ). HCV NS3 represents HCV infection and actin was used as protein loading control. (C) Immunofluorescence microscopy of HCV-infected PHH. HCV-infected PHH was incubated with HCV NS5A antibody for 1 h at RT followed by 1 h incubation with secondary HCV NS5A antibody (anti-rabbit Alexa Fluor 546) as describe in Materials and Methods. The magenta color indicates expression of HCV NS5A around the nucleus. Arrow indicates few uninfected cells. DAPI was used as a nuclear stain in blue color. Scale bar 10 µM. The results shown are the representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes

    doi: 10.1371/journal.pone.0087464

    Figure Lengend Snippet: HCV induces OPN and signaling cascade in primary human hepatocytes (PHH). (A, B) Equal amounts of cellular lysates from PHH (lane 1) and HCV-infected PHH (lane 2) were subjected to western blot analysis using anti-OPN, anti-CD44, anti-β3, anti-E-cadherin, anti-N-cadherin, anti-FAK, and anti-pAkt (Ser 473 ). HCV NS3 represents HCV infection and actin was used as protein loading control. (C) Immunofluorescence microscopy of HCV-infected PHH. HCV-infected PHH was incubated with HCV NS5A antibody for 1 h at RT followed by 1 h incubation with secondary HCV NS5A antibody (anti-rabbit Alexa Fluor 546) as describe in Materials and Methods. The magenta color indicates expression of HCV NS5A around the nucleus. Arrow indicates few uninfected cells. DAPI was used as a nuclear stain in blue color. Scale bar 10 µM. The results shown are the representative of three independent experiments.

    Article Snippet: The results show significant colocalization of OPN with pan-cadherin in permeabilized ( ) and nonpermeabilized HCV-infected cells ( ).

    Techniques: Infection, Western Blot, Immunofluorescence, Microscopy, Incubation, Expressing, Staining

    Role of HCV-induced OPN in cell signaling cascade. (A) HCV-infected cells were transfected with siGFP, siOPN, siCD44, and siβ3 as described in Materials and Methods. At 72 h posttransfection, cells were harvested and equal amount of cellular lysates were subjected to western blot analysis using anti-OPN, anti-β3 and anti-CD44. (B) The above cellular lysates were subjected to western blot analysis using anti-FAK, anti-p-Akt (Ser 473 ), anti-p-Src (Tyr 416 ), and anti-N-cadherin antibodies. HCV NS3 was used as a representative of HCV-infection. (C) Mock (Huh7.5) cells were treated with recombinant human OPN (rhOPN) (50 nM) for 48 h. Cells were harvested and equal amounts of cellular lysates were immunoblotted using anti-FAK and anti-p-Akt (Ser 473 ) (lane 1, 2), anti-E-cadherin, anti-N-cadherin, anti-pSrc 416 (lane 3, 4). (D) Cellular lysates from HepG2 cells incubated with cell culture supernatants from mock, HCV-infected cells and those infected cells transfected with siOPN and siGFP were immunoblotted using anti-E-cadherin and anti-N-cadherin (lane 1–4). Similarly cellular lysates from HepG2 cells incubated with cell culture supernatants from mock and HCV-infected cells with/without immunodepletion by anti-OPN, were immunoblotted using anti-E-cadherin and anti-N-cadherin (lane 5–7). Immunodepletion by isotype control goat IgG antibody was used as control (lane 8). Actin was used as protein loading control. The results shown are the representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes

    doi: 10.1371/journal.pone.0087464

    Figure Lengend Snippet: Role of HCV-induced OPN in cell signaling cascade. (A) HCV-infected cells were transfected with siGFP, siOPN, siCD44, and siβ3 as described in Materials and Methods. At 72 h posttransfection, cells were harvested and equal amount of cellular lysates were subjected to western blot analysis using anti-OPN, anti-β3 and anti-CD44. (B) The above cellular lysates were subjected to western blot analysis using anti-FAK, anti-p-Akt (Ser 473 ), anti-p-Src (Tyr 416 ), and anti-N-cadherin antibodies. HCV NS3 was used as a representative of HCV-infection. (C) Mock (Huh7.5) cells were treated with recombinant human OPN (rhOPN) (50 nM) for 48 h. Cells were harvested and equal amounts of cellular lysates were immunoblotted using anti-FAK and anti-p-Akt (Ser 473 ) (lane 1, 2), anti-E-cadherin, anti-N-cadherin, anti-pSrc 416 (lane 3, 4). (D) Cellular lysates from HepG2 cells incubated with cell culture supernatants from mock, HCV-infected cells and those infected cells transfected with siOPN and siGFP were immunoblotted using anti-E-cadherin and anti-N-cadherin (lane 1–4). Similarly cellular lysates from HepG2 cells incubated with cell culture supernatants from mock and HCV-infected cells with/without immunodepletion by anti-OPN, were immunoblotted using anti-E-cadherin and anti-N-cadherin (lane 5–7). Immunodepletion by isotype control goat IgG antibody was used as control (lane 8). Actin was used as protein loading control. The results shown are the representative of three independent experiments.

    Article Snippet: The results show significant colocalization of OPN with pan-cadherin in permeabilized ( ) and nonpermeabilized HCV-infected cells ( ).

    Techniques: Infection, Transfection, Western Blot, Recombinant, Incubation, Cell Culture

    Colocalization of OPN with integrin αVβ3 and CD44. (A, B) Mock and HCV-infected cells (from figure 2A ) were transfected with siGFP and siOPN. At 72 h posttransfection, cells were permeabilized and incubated with anti-OPN, anti-αVβ3, anti-CD44 and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), αVβ3 (anti-mouse Alexa Fluor 488), CD44 (anti-mouse Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with αVβ3 and CD44 respectively. HCV NS5A represents HCV infection. Scale bar 10 µM. (C) Colocalization of OPN with pan-cadherin (plasma membrane marker). Mock and HCV-infected cells (from figure 2A ) were permeabilized and incubated with anti-OPN, anti-pan-cadherin and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), pan-cadherin (anti-rabbit Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). (D) Similarly, non-permeabilized mock and HCV-infected cells were incubated with anti-OPN and anti-pan-cadherin antibodies for 1 h at RT and then cells were permeabilized and incubated with anti-HCV NS5A antibody for 1 h at RT followed by 1 h incubation with above secondary antibodies. DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with pan-cadherin. (E) Colocalization of OPN with PDI (ER marker). As described in panel C and D, permeabilized cells were incubated with anti-OPN, anti-PDI and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), PDI (anti-rabbit Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). (F) Simultaneously, non-permeabilized cells were incubated with anti-OPN and anti-PDI antibodies for 1 h at RT and then cells were permeabilized and incubated with anti-HCV NS5A antibody for 1 h at RT followed by 1 h incubation with above secondary antibodies. DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with ER marker. HCV NS5A represents HCV infection. Scale bar 10 µM.

    Journal: PLoS ONE

    Article Title: Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes

    doi: 10.1371/journal.pone.0087464

    Figure Lengend Snippet: Colocalization of OPN with integrin αVβ3 and CD44. (A, B) Mock and HCV-infected cells (from figure 2A ) were transfected with siGFP and siOPN. At 72 h posttransfection, cells were permeabilized and incubated with anti-OPN, anti-αVβ3, anti-CD44 and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), αVβ3 (anti-mouse Alexa Fluor 488), CD44 (anti-mouse Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with αVβ3 and CD44 respectively. HCV NS5A represents HCV infection. Scale bar 10 µM. (C) Colocalization of OPN with pan-cadherin (plasma membrane marker). Mock and HCV-infected cells (from figure 2A ) were permeabilized and incubated with anti-OPN, anti-pan-cadherin and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), pan-cadherin (anti-rabbit Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). (D) Similarly, non-permeabilized mock and HCV-infected cells were incubated with anti-OPN and anti-pan-cadherin antibodies for 1 h at RT and then cells were permeabilized and incubated with anti-HCV NS5A antibody for 1 h at RT followed by 1 h incubation with above secondary antibodies. DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with pan-cadherin. (E) Colocalization of OPN with PDI (ER marker). As described in panel C and D, permeabilized cells were incubated with anti-OPN, anti-PDI and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), PDI (anti-rabbit Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). (F) Simultaneously, non-permeabilized cells were incubated with anti-OPN and anti-PDI antibodies for 1 h at RT and then cells were permeabilized and incubated with anti-HCV NS5A antibody for 1 h at RT followed by 1 h incubation with above secondary antibodies. DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with ER marker. HCV NS5A represents HCV infection. Scale bar 10 µM.

    Article Snippet: The results show significant colocalization of OPN with pan-cadherin in permeabilized ( ) and nonpermeabilized HCV-infected cells ( ).

    Techniques: Infection, Transfection, Incubation, Staining, Marker

    HCV induces EMT via OPN. HCV-infected cells were transfected with siOPN and siGFP as described in Materials and Methods. At 72 h posttransfection, cells were harvested and equal amounts of cellular lysates were immunoblotted with anti-E-cadherin (A), anti-N-cadherin (B), and anti-OPN (A, B). Actin was used as protein loading control.

    Journal: PLoS ONE

    Article Title: Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes

    doi: 10.1371/journal.pone.0087464

    Figure Lengend Snippet: HCV induces EMT via OPN. HCV-infected cells were transfected with siOPN and siGFP as described in Materials and Methods. At 72 h posttransfection, cells were harvested and equal amounts of cellular lysates were immunoblotted with anti-E-cadherin (A), anti-N-cadherin (B), and anti-OPN (A, B). Actin was used as protein loading control.

    Article Snippet: The results show significant colocalization of OPN with pan-cadherin in permeabilized ( ) and nonpermeabilized HCV-infected cells ( ).

    Techniques: Infection, Transfection

    Development of isogenic SYNGAP1 knockout hiPSCs. (A ) Cartoon showing clone-specific mutations in the SYNGAP1 gene. (B) Sanger sequencing for one WT clone and two SYNGAP1 mutant clones derived from the CRISPR experiment. ( C ) Whole genome view of iNeurons from WT#6, WT#30, KO#4 and KO#38 clones depicting a copy number value of 2 cross all chromosomes (expect for the Y-chromosome which is not detected) revealing normal (female) karyotype with no chromosomal aberrations. The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number and aberrations (if any). ( D ) Western blots demonstrating SynGAP protein expression from iNeuron or iPSC homogenate. Total refers to signal from an antibody that detects all splice variants and α2 refers to signal from an antibody that detects only a specific C-terminal splice variant. ( E ) Quantification of relative intensity of bands normalized to total protein signal. One-way ANOVA with a Kruskal-Wallis test multiple comparisons test H(3)=12.29, p=0.0001; WT#6 vs KO#4: p=0.1876; WT#6 vs WT#30: p > 0.9999; WT#6 vs KO#38: p=0.0140; KO#4 vs WT#30: p=0.5258; KO#4 vs KO#38: p > 0.9999; WT#30 vs KO#38: p=0.0561. n=4 per group. In box-and-whisker plot, the center, boxes and whiskers represent the median, interquartile range, and min to max, respectively. (F) Indels from each clone identified from whole exome sequencing analysis. Indels were identified by clonal sequence differences from the original Cas9 hiPSCs (reference sequence). Indel threshold was determined by at least 50% of the reads differing from the reference sequence with a minimum of at least ten reads. Indels w/ frequency above 0.8 were used to determine frequency of homozygous varaints. ( G ) Normalized mapped reads from the entire coding sequence of the SYNGAP1 gene in the four clones hiPSCs. Red arrow denotes predicted Cas9 cut site. Numbers reflect clonal reads relative to Cas9 hiPSC reads. ( H ) Normalized mapped reads for the same samples around the Cas9 target sequence. ( I ) Genomic PCR to amplify DNA sequence flanking the Cas9 target site.

    Journal: bioRxiv

    Article Title: Human SYNGAP1 Regulates the Development of Neuronal Activity by Controlling Dendritic and Synaptic Maturation

    doi: 10.1101/2020.06.01.127613

    Figure Lengend Snippet: Development of isogenic SYNGAP1 knockout hiPSCs. (A ) Cartoon showing clone-specific mutations in the SYNGAP1 gene. (B) Sanger sequencing for one WT clone and two SYNGAP1 mutant clones derived from the CRISPR experiment. ( C ) Whole genome view of iNeurons from WT#6, WT#30, KO#4 and KO#38 clones depicting a copy number value of 2 cross all chromosomes (expect for the Y-chromosome which is not detected) revealing normal (female) karyotype with no chromosomal aberrations. The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number and aberrations (if any). ( D ) Western blots demonstrating SynGAP protein expression from iNeuron or iPSC homogenate. Total refers to signal from an antibody that detects all splice variants and α2 refers to signal from an antibody that detects only a specific C-terminal splice variant. ( E ) Quantification of relative intensity of bands normalized to total protein signal. One-way ANOVA with a Kruskal-Wallis test multiple comparisons test H(3)=12.29, p=0.0001; WT#6 vs KO#4: p=0.1876; WT#6 vs WT#30: p > 0.9999; WT#6 vs KO#38: p=0.0140; KO#4 vs WT#30: p=0.5258; KO#4 vs KO#38: p > 0.9999; WT#30 vs KO#38: p=0.0561. n=4 per group. In box-and-whisker plot, the center, boxes and whiskers represent the median, interquartile range, and min to max, respectively. (F) Indels from each clone identified from whole exome sequencing analysis. Indels were identified by clonal sequence differences from the original Cas9 hiPSCs (reference sequence). Indel threshold was determined by at least 50% of the reads differing from the reference sequence with a minimum of at least ten reads. Indels w/ frequency above 0.8 were used to determine frequency of homozygous varaints. ( G ) Normalized mapped reads from the entire coding sequence of the SYNGAP1 gene in the four clones hiPSCs. Red arrow denotes predicted Cas9 cut site. Numbers reflect clonal reads relative to Cas9 hiPSC reads. ( H ) Normalized mapped reads for the same samples around the Cas9 target sequence. ( I ) Genomic PCR to amplify DNA sequence flanking the Cas9 target site.

    Article Snippet: Membranes were blocked with 5% powdered milk in buffer and probed with pan-SynGAP (1:1,000, #5539, Cell Signaling) or SynGAP-α2 (abcam, ab77235), overnight at 4°C and HRP-conjugated anti-rabbit antibody (1:2,000, W4011, Promega) for 1 hr at room temperature followed by ECL signal amplification and chemiluminescence detection (SuperSignal West Pico Chemiluminescent Substrate; Thermo Scientific, Rockford, IL).

    Techniques: Knock-Out, Sequencing, Mutagenesis, Clone Assay, Derivative Assay, CRISPR, Western Blot, Expressing, Variant Assay, Whisker Assay, Polymerase Chain Reaction

    Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

    Journal: PLoS ONE

    Article Title: Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

    doi: 10.1371/journal.pone.0089352

    Figure Lengend Snippet: Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

    Article Snippet: The primary antibodies (for immunostaining and some also for western blotting) were: rabbit anti-FlnA monoclonal antibody (1∶300, Cat.# 2242, Epitomics, Burlingame, CA, USA); rabbit anti-FlnB polyclonal antibody (Gifted by Dr. Kao, CWRU); mouse anti-Col2a1 (Cat.# Ab3092, ABCAM, USA); rabbit anti-Col10a1 (kindly gifted by Dr. Horton and Dr. Lunstrum, Shriners Hospital for Children, Portland, OR, USA; ); rabbit anti-Pthr1 (Cat.# Ab75150, ABCAM, USA);rabbit anti-Ihh (Cat.# sc-13088, Santa Cruz); rabbit anti-Runx2 (Cat.# sc-10758, Santa Cruz); rabbit anti-Sox9 pab (1∶300, AB5535, Millipore); rabbit anti-Sox9 pab (O9-1, gift of Professor Dr. Michael Wegner, Institute of Biochemistry, Friedrich-Alexander-University, Erlangen-Nurnberg, Germany); rat anti-BrdU (1∶150, Cat.# MCA2060, AbD Serotec, Raleigh, NC, USA); rabbit anti-Ki-67 mab (1∶200, Cat.# 4203, Epitomics); rabbit anti-PH3 pab (1∶250, Cat:# 06-570, Millipore, Billerica, MA, USA); mouse and rabbit anti-Wee1 (1∶50, Cat.# sc-5285 and sc-325, Santa Cruz); rabbit anti-Pkmyt1 (1∶100, Cat.# 3303, Epitomics); anti-pan 14-3-3 (Santa Cruz, sc-629); mouse and rabbit anti-cyclin B1 (1∶100, Cat.# sc-245 and sc-752, Santa Cruz); mouse anti-Cdc20 (1∶100, Cat.# sc-13162, Santa Cruz); mouse and rabbit anti-cdc25c (1∶100, Cat.# sc-55513 and sc-327, Santa Cruz); rabbit-anti-Cdk1 (Cat.# PC25,Calbiochem, San Diego, CA, USA); mouse anti-Cdk1(pY15) (Cat.# BD612306, BD, Franklin Lakes, NJ USA); rabbit anti-Pi3k (p85 subunit alpha, Cat#: 1675, Epitomics); rabbit anti-Akt (phospho-S473, Cat# 4060, Cell Signaling); rabbit anti-Erk1/2 (phospho-T202/Y204, Cat.# 4370, Cell Signaling); rat anti-β1 integrin (Cat.# mab1997, Millipore); rabbit anti- β1 integrin(pS785) (Cat.# OPA1-03177, Affinity BioReagents).

    Techniques: Activity Assay, Immunostaining, Knock-Out, Western Blot, Activation Assay, Incubation, Laser Capture Microdissection