β-actin Search Results


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  • 90
    Thermo Fisher β actin mn00607939 s1
    β Actin Mn00607939 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti βactin
    ( A,C ) Changes in the expression levels of matrix metalloproteinases (MMPs) following treatment with 28-hydroxy-3-oxoolean-12-en-29-oic acid for 24 h were assessed by Western blotting. ( B,D ) The band intensities of TIMP-1,MMP-9 and MMP-2 relative to untreated control cells were quantified upon normalizing to <t>β-actin</t> expression, and are expressed as the mean ± standard deviation of three independent experiments.
    Anti βactin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6453 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti βactin/product/Cell Signaling Technology Inc
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    Cell Signaling Technology Inc anti β actin
    Expression of other IFT proteins in conditional Ift140 KO mice A. Representative Western blotting images showing testicular expression of IFT20, IFT25, IFT27, IFT74, IFT81, and IFT88 using specific antibodies in three control and three conditional Ift140 mice. <t>β-actin</t> was used as a loading control. B. Statistical analysis of relative expression of the IFT proteins normalized by β-actin. There is no difference in expression levels of these selective IFT proteins between the controls and the Ift140 KO mice. C. Localization of IFT20 in the germ cells of control and conditional Ift140 knockout mice. In mice with both genotypes, IFT20 is present in the Golgi bodies of spermatocytes (upper panels), acrosome of round spermatids (middle panels), and manchette of elongating spermatids (lower panels). However, in the control animal, IFT20 appears to attach closely to the nuclear membrane (white arrow) in round spermatids; in the Ift140 KO, IFT20 does not seem to attach tightly to the nuclear membrane (dashed white arrow). D. Abnormal localization of IFT27 in the conditional Ift140 knockout mice. The granule-like pattern of the IFT27 signal was never observed in the control mice. E. Abnormal localization of IFT88 in the conditional Ift140 knockout mice. In the control mouse, IFT88 is present in the cytoplasm of spermatocytes and round spermatids, manchette of elongating spermatids, and the developing tails; In the Ift140 KO mice, even though IFT88 is still present in the cytoplasm of spermatocytes and round spermatids, it accumulates abnormally in the germ cells at later stages (white arrow heads).
    Anti β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Proteintech β‑actin
    Expression of other IFT proteins in conditional Ift140 KO mice A. Representative Western blotting images showing testicular expression of IFT20, IFT25, IFT27, IFT74, IFT81, and IFT88 using specific antibodies in three control and three conditional Ift140 mice. <t>β-actin</t> was used as a loading control. B. Statistical analysis of relative expression of the IFT proteins normalized by β-actin. There is no difference in expression levels of these selective IFT proteins between the controls and the Ift140 KO mice. C. Localization of IFT20 in the germ cells of control and conditional Ift140 knockout mice. In mice with both genotypes, IFT20 is present in the Golgi bodies of spermatocytes (upper panels), acrosome of round spermatids (middle panels), and manchette of elongating spermatids (lower panels). However, in the control animal, IFT20 appears to attach closely to the nuclear membrane (white arrow) in round spermatids; in the Ift140 KO, IFT20 does not seem to attach tightly to the nuclear membrane (dashed white arrow). D. Abnormal localization of IFT27 in the conditional Ift140 knockout mice. The granule-like pattern of the IFT27 signal was never observed in the control mice. E. Abnormal localization of IFT88 in the conditional Ift140 knockout mice. In the control mouse, IFT88 is present in the cytoplasm of spermatocytes and round spermatids, manchette of elongating spermatids, and the developing tails; In the Ift140 KO mice, even though IFT88 is still present in the cytoplasm of spermatocytes and round spermatids, it accumulates abnormally in the germ cells at later stages (white arrow heads).
    β‑Actin, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β actin protein
    HBxΔ127 has a greater capacity to stimulate SREBP-1c relative to wild type HBx. (A, B) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were transfected with pSilencer 3.0-X plasmid for 48 h. (A) The mRNA levels of SREBP-1c were detected by RT-PCR. (B) Immunoblot analysis to detect SREBP-1c (top panel) and <t>β-actin</t> protein levels (bottom panel). (C, D) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were co-transfected with or without pSilencer 3.0-X plasmid and SREBP-1c-571-Luc-WT. Data are representative of 3 independent experiments. Values represent means±SD. n =3. b P
    β Actin Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc polyclonal anti β actin
    HBxΔ127 has a greater capacity to stimulate SREBP-1c relative to wild type HBx. (A, B) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were transfected with pSilencer 3.0-X plasmid for 48 h. (A) The mRNA levels of SREBP-1c were detected by RT-PCR. (B) Immunoblot analysis to detect SREBP-1c (top panel) and <t>β-actin</t> protein levels (bottom panel). (C, D) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were co-transfected with or without pSilencer 3.0-X plasmid and SREBP-1c-571-Luc-WT. Data are representative of 3 independent experiments. Values represent means±SD. n =3. b P
    Polyclonal Anti β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti βactin
    HBxΔ127 has a greater capacity to stimulate SREBP-1c relative to wild type HBx. (A, B) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were transfected with pSilencer 3.0-X plasmid for 48 h. (A) The mRNA levels of SREBP-1c were detected by RT-PCR. (B) Immunoblot analysis to detect SREBP-1c (top panel) and <t>β-actin</t> protein levels (bottom panel). (C, D) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were co-transfected with or without pSilencer 3.0-X plasmid and SREBP-1c-571-Luc-WT. Data are representative of 3 independent experiments. Values represent means±SD. n =3. b P
    Rabbit Anti βactin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti βactin/product/Cell Signaling Technology Inc
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    Image Search Results


    ( A,C ) Changes in the expression levels of matrix metalloproteinases (MMPs) following treatment with 28-hydroxy-3-oxoolean-12-en-29-oic acid for 24 h were assessed by Western blotting. ( B,D ) The band intensities of TIMP-1,MMP-9 and MMP-2 relative to untreated control cells were quantified upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments.

    Journal: Molecules

    Article Title: 28-Hydroxy-3-oxoolean-12-en-29-oic Acid, a Triterpene Acid from Celastrus Orbiculatus Extract, Inhibits the Migration and Invasion of Human Gastric Cancer Cells In Vitro

    doi: 10.3390/molecules24193513

    Figure Lengend Snippet: ( A,C ) Changes in the expression levels of matrix metalloproteinases (MMPs) following treatment with 28-hydroxy-3-oxoolean-12-en-29-oic acid for 24 h were assessed by Western blotting. ( B,D ) The band intensities of TIMP-1,MMP-9 and MMP-2 relative to untreated control cells were quantified upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments.

    Article Snippet: Reagents The reagents used included the following: RPMI 1640 cell culture medium, trypsin (HyClone, USA); fetal bovine serum (FBS) (Gibco, Waltham, MA, USA); artificially reconstituted basement membrane, Transwell chambers (Corning, Corning, NY, USA); thiazole blue (MTT) powder (Sigma, St. Louis, MO, USA); antibodies specific for E-cadherin, N-cadherin, vimentin, Snail, MMP-2, MMP-9, Akt, p-AKt, PI3K, p-PI3K, and β-actin (β-actin) (Cell Signaling, Danvers, MA, USA); and HRP-labeled goat anti-rabbit immunoglobulin (Ig) G (Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China).

    Techniques: Expressing, Western Blot, Standard Deviation

    ( A , C ) Changes in PI3K/AKt/Snail biomarker expression levels following treatment with 28-hydroxy-3-oxoolean-12-en-29-oic acid for 24 h were assessed by Western blotting. ( B , D ) The band intensities of AKt, (p)-Akt, PI3K, (p)-PI3K and Snail relative to untreated control cells were quantified upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments.

    Journal: Molecules

    Article Title: 28-Hydroxy-3-oxoolean-12-en-29-oic Acid, a Triterpene Acid from Celastrus Orbiculatus Extract, Inhibits the Migration and Invasion of Human Gastric Cancer Cells In Vitro

    doi: 10.3390/molecules24193513

    Figure Lengend Snippet: ( A , C ) Changes in PI3K/AKt/Snail biomarker expression levels following treatment with 28-hydroxy-3-oxoolean-12-en-29-oic acid for 24 h were assessed by Western blotting. ( B , D ) The band intensities of AKt, (p)-Akt, PI3K, (p)-PI3K and Snail relative to untreated control cells were quantified upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments.

    Article Snippet: Reagents The reagents used included the following: RPMI 1640 cell culture medium, trypsin (HyClone, USA); fetal bovine serum (FBS) (Gibco, Waltham, MA, USA); artificially reconstituted basement membrane, Transwell chambers (Corning, Corning, NY, USA); thiazole blue (MTT) powder (Sigma, St. Louis, MO, USA); antibodies specific for E-cadherin, N-cadherin, vimentin, Snail, MMP-2, MMP-9, Akt, p-AKt, PI3K, p-PI3K, and β-actin (β-actin) (Cell Signaling, Danvers, MA, USA); and HRP-labeled goat anti-rabbit immunoglobulin (Ig) G (Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China).

    Techniques: Biomarker Assay, Expressing, Western Blot, Standard Deviation

    ( A , C ) Changes in epithelial–mesenchymal transition (EMT) biomarker expression levels following 28-hydroxy-3-oxoolean-12-en-29-oic acid for 24 h were assessed by Western blotting. ( B , D ) The band intensities of E-cadherin, N-cadherin and vimentin relative to untreated control cells were quantified upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments.

    Journal: Molecules

    Article Title: 28-Hydroxy-3-oxoolean-12-en-29-oic Acid, a Triterpene Acid from Celastrus Orbiculatus Extract, Inhibits the Migration and Invasion of Human Gastric Cancer Cells In Vitro

    doi: 10.3390/molecules24193513

    Figure Lengend Snippet: ( A , C ) Changes in epithelial–mesenchymal transition (EMT) biomarker expression levels following 28-hydroxy-3-oxoolean-12-en-29-oic acid for 24 h were assessed by Western blotting. ( B , D ) The band intensities of E-cadherin, N-cadherin and vimentin relative to untreated control cells were quantified upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments.

    Article Snippet: Reagents The reagents used included the following: RPMI 1640 cell culture medium, trypsin (HyClone, USA); fetal bovine serum (FBS) (Gibco, Waltham, MA, USA); artificially reconstituted basement membrane, Transwell chambers (Corning, Corning, NY, USA); thiazole blue (MTT) powder (Sigma, St. Louis, MO, USA); antibodies specific for E-cadherin, N-cadherin, vimentin, Snail, MMP-2, MMP-9, Akt, p-AKt, PI3K, p-PI3K, and β-actin (β-actin) (Cell Signaling, Danvers, MA, USA); and HRP-labeled goat anti-rabbit immunoglobulin (Ig) G (Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China).

    Techniques: Biomarker Assay, Expressing, Western Blot, Standard Deviation

    4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and β -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

    doi: 10.1155/2018/2747018

    Figure Lengend Snippet: 4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and β -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p

    Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

    Techniques: Expressing, MTT Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Quantitative RT-PCR

    4-PG prevents LPS-induced acute lung injury and upregulates HO-1 expression. (a) The scheme depicts the experimental protocol used to assess the protective effect of pterostilbene 4′-glucoside (4-PG) and pterostilbene (PTER) on LPS-induced ALI. 10-week-old mice were injected with 4-PG (10 mg/kg, i.p.) and PTER (10 mg/kg, i.p.) for 4 days prior to intranasal administration of LPS (2.5 mg/kg) for 24 h. (b) Chemical structures of 4-PG and PTER. (c) Lung sections were stained with hematoxylin and eosin (H E) for morphological evaluation, and the representative lung sections of each group are shown. Scale bar = 100 μ m. (left). Quantitative analysis of histologic lung section by lung injury score for six experimental groups. The score generates the average of two independent investigators (right). (d, e) The mRNA expression of proinflammatory cytokines and chemokines (TNF- α , IL-6, IL-1 β , CXCL1, and CXCL2) in lung tissues was detected by RT-PCR. Furthermore, mRNA and protein levels of HO-1 were assessed by RT-PCR (f, left: HO-1mRNA levels, right: quantification of the relative band density) and Western blotting (g, left: HO-1 protein levels, right: quantification of the relative band density) from lung tissues, respectively. 18S and β -actin were used as internal controls. Data were expressed as mean ± SD ( n = 5 per group); ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

    doi: 10.1155/2018/2747018

    Figure Lengend Snippet: 4-PG prevents LPS-induced acute lung injury and upregulates HO-1 expression. (a) The scheme depicts the experimental protocol used to assess the protective effect of pterostilbene 4′-glucoside (4-PG) and pterostilbene (PTER) on LPS-induced ALI. 10-week-old mice were injected with 4-PG (10 mg/kg, i.p.) and PTER (10 mg/kg, i.p.) for 4 days prior to intranasal administration of LPS (2.5 mg/kg) for 24 h. (b) Chemical structures of 4-PG and PTER. (c) Lung sections were stained with hematoxylin and eosin (H E) for morphological evaluation, and the representative lung sections of each group are shown. Scale bar = 100 μ m. (left). Quantitative analysis of histologic lung section by lung injury score for six experimental groups. The score generates the average of two independent investigators (right). (d, e) The mRNA expression of proinflammatory cytokines and chemokines (TNF- α , IL-6, IL-1 β , CXCL1, and CXCL2) in lung tissues was detected by RT-PCR. Furthermore, mRNA and protein levels of HO-1 were assessed by RT-PCR (f, left: HO-1mRNA levels, right: quantification of the relative band density) and Western blotting (g, left: HO-1 protein levels, right: quantification of the relative band density) from lung tissues, respectively. 18S and β -actin were used as internal controls. Data were expressed as mean ± SD ( n = 5 per group); ∗∗ p

    Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

    Techniques: Expressing, Mouse Assay, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Triple-drug (LY294002, PLX4720 and lexatumumab) and dual-drug combinations (LY294002 and lexatumumab) triggered the intrinsic and extrinsic apoptotic pathways in 8505c and SW1736 cells. 8505c and SW1736 cells were treated for 8 h with drug combinations as indicated in the figure and analyzed for pro- and anti-apoptotic proteins by western blot. ( a ) Treatment with triple-drug combination in 8505c and the dual combination in SW1736 resulted in cleavage of caspase 8/9/3 and PARP. ( b ) Triple-drug combination resulted in the increase in pro-apoptotic proteins Bad, Bim, Bax and Bak in 8505c cells. Dual inhibitor combination, on the other hand, resulted in an increase in BimL  S forms in SW1736 cells. ( c ) Triple-drug combination significantly decreased the anti-apoptotic proteins Bcl-xL and Mcl-1 in 8505c cells at 8 h of treatment. ( d ) cFLIP decreased in both the dual and triple-drug combinational treatments of 8505c cells. ( e ) Treatment of 8505c cells at different time points up to 48 h with the triple-drug combination also showed a significant decrease in anti- apoptotic proteins, Bcl-xL, Mcl-1, cFlip and an increase in pro-apoptotic proteins, Bax and Bim isoforms.  β -Actin was used as loading control

    Journal: Cell Death & Disease

    Article Title: Blocks to thyroid cancer cell apoptosis can be overcome by inhibition of the MAPK and PI3K/AKT pathways

    doi: 10.1038/cddis.2014.78

    Figure Lengend Snippet: Triple-drug (LY294002, PLX4720 and lexatumumab) and dual-drug combinations (LY294002 and lexatumumab) triggered the intrinsic and extrinsic apoptotic pathways in 8505c and SW1736 cells. 8505c and SW1736 cells were treated for 8 h with drug combinations as indicated in the figure and analyzed for pro- and anti-apoptotic proteins by western blot. ( a ) Treatment with triple-drug combination in 8505c and the dual combination in SW1736 resulted in cleavage of caspase 8/9/3 and PARP. ( b ) Triple-drug combination resulted in the increase in pro-apoptotic proteins Bad, Bim, Bax and Bak in 8505c cells. Dual inhibitor combination, on the other hand, resulted in an increase in BimL S forms in SW1736 cells. ( c ) Triple-drug combination significantly decreased the anti-apoptotic proteins Bcl-xL and Mcl-1 in 8505c cells at 8 h of treatment. ( d ) cFLIP decreased in both the dual and triple-drug combinational treatments of 8505c cells. ( e ) Treatment of 8505c cells at different time points up to 48 h with the triple-drug combination also showed a significant decrease in anti- apoptotic proteins, Bcl-xL, Mcl-1, cFlip and an increase in pro-apoptotic proteins, Bax and Bim isoforms. β -Actin was used as loading control

    Article Snippet: Antibodies – β -actin, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (phospho-ERK), p44/42 MAPK (ERK1/2) (Total ERK), total Akt, phospho-Akt (Ser473 ), caspases 3,8,9, cleaved caspase-3, PARP, c-FLIP, TRAIL-R2, Bcl2, Bcl-xL, Mcl-1, Bim, Bid, Bax, Bad, Bak were purchased from Cell Signaling Technology.

    Techniques: Western Blot

    Knockdown of Bcl-xL sensitized 8505c cells to lexatumumab-induced apoptosis. Cells were transfected with 50-nM Bcl-xL or control siRNA for 48 h and then treated with 1000 ng/ml of lexatumumab for another 24 h. ( a ) Bcl-xL protein level significantly decreased after 48 h of transfection with Bcl-xL siRNA.  β -Actin served as a loading control. ( b ) Treatment of Bcl-xL knockdown cells with lexatumumab showed a significant increase in cell death (52.3%,  P

    Journal: Cell Death & Disease

    Article Title: Blocks to thyroid cancer cell apoptosis can be overcome by inhibition of the MAPK and PI3K/AKT pathways

    doi: 10.1038/cddis.2014.78

    Figure Lengend Snippet: Knockdown of Bcl-xL sensitized 8505c cells to lexatumumab-induced apoptosis. Cells were transfected with 50-nM Bcl-xL or control siRNA for 48 h and then treated with 1000 ng/ml of lexatumumab for another 24 h. ( a ) Bcl-xL protein level significantly decreased after 48 h of transfection with Bcl-xL siRNA. β -Actin served as a loading control. ( b ) Treatment of Bcl-xL knockdown cells with lexatumumab showed a significant increase in cell death (52.3%, P

    Article Snippet: Antibodies – β -actin, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (phospho-ERK), p44/42 MAPK (ERK1/2) (Total ERK), total Akt, phospho-Akt (Ser473 ), caspases 3,8,9, cleaved caspase-3, PARP, c-FLIP, TRAIL-R2, Bcl2, Bcl-xL, Mcl-1, Bim, Bid, Bax, Bad, Bak were purchased from Cell Signaling Technology.

    Techniques: Transfection

    TAM tyrosine kinase receptors are novel ubiquitylation targets of Cbl-b. a, Out of 9000 human proteins tested, Tyro3 had the highest Cbl-b mediated ubiquitylation signal. Signal intensities are shown for the corresponding Tyro3 spots in the protein arrays incubated with the E2 enzyme without Cbl-b, the Cbl-b C373A mutant, and wild-type Cbl-b proteins (in duplicates). Data are shown as mean values. RFU: relative fluorescent units. b, In vitro ubiquitylation of recombinant Flag-tagged Tyro3, Axl, and Mer in the presence (left panel) and absence (right panel) of Cbl-b. Blots were probed with anti-Flag Abs. c, Immunoprecipitation showing time-dependent recruitment of Cbl-b to TAM tyrosine kinase receptors in HeLa cells upon stimulation with His-tagged Gas6 (upper panel). Input levels of Cbl-b and β-actin are shown as controls. d , Gas6-induced Axl ubiquitylation depends on Cbl-b expression. HeLa cells were transfected with scrambled siControl or siCbl-b and then stimulated with 450ng/ml Gas6 for 15 minutes or left untreated. Lysates were immunoprecipiated with anti-Axl antibodies. Blots were then probed using anti-Ubiquitin and anti-Axl Abs. The location of the mature form of Axl (~140kDa) is shown with an arrow. Axl, Cbl-b, and tubulin protein levels are shown to control for the input (lower panels). e, Representative histograms showing equal expression of Tyro3, Axl, and Mer receptors at the cell surface of freshly isolated NK1.1 + CD3ε - splenic Cbl-b +/+ Cbl-b -/- and C373A KI/KI NK cells. Background staining with control isotype Abs is shown for each blot in grey. f, In vitro proliferation of Cbl-b +/- and Cbl-b -/- NK cells stimulated with anti-NKG2D Abs (30μg/ml) in the presence of different concentrations of Gas6 (mean values ± s.e.m., n=4). *P

    Journal: Nature

    Article Title: The E3 Ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells

    doi: 10.1038/nature12998

    Figure Lengend Snippet: TAM tyrosine kinase receptors are novel ubiquitylation targets of Cbl-b. a, Out of 9000 human proteins tested, Tyro3 had the highest Cbl-b mediated ubiquitylation signal. Signal intensities are shown for the corresponding Tyro3 spots in the protein arrays incubated with the E2 enzyme without Cbl-b, the Cbl-b C373A mutant, and wild-type Cbl-b proteins (in duplicates). Data are shown as mean values. RFU: relative fluorescent units. b, In vitro ubiquitylation of recombinant Flag-tagged Tyro3, Axl, and Mer in the presence (left panel) and absence (right panel) of Cbl-b. Blots were probed with anti-Flag Abs. c, Immunoprecipitation showing time-dependent recruitment of Cbl-b to TAM tyrosine kinase receptors in HeLa cells upon stimulation with His-tagged Gas6 (upper panel). Input levels of Cbl-b and β-actin are shown as controls. d , Gas6-induced Axl ubiquitylation depends on Cbl-b expression. HeLa cells were transfected with scrambled siControl or siCbl-b and then stimulated with 450ng/ml Gas6 for 15 minutes or left untreated. Lysates were immunoprecipiated with anti-Axl antibodies. Blots were then probed using anti-Ubiquitin and anti-Axl Abs. The location of the mature form of Axl (~140kDa) is shown with an arrow. Axl, Cbl-b, and tubulin protein levels are shown to control for the input (lower panels). e, Representative histograms showing equal expression of Tyro3, Axl, and Mer receptors at the cell surface of freshly isolated NK1.1 + CD3ε - splenic Cbl-b +/+ Cbl-b -/- and C373A KI/KI NK cells. Background staining with control isotype Abs is shown for each blot in grey. f, In vitro proliferation of Cbl-b +/- and Cbl-b -/- NK cells stimulated with anti-NKG2D Abs (30μg/ml) in the presence of different concentrations of Gas6 (mean values ± s.e.m., n=4). *P

    Article Snippet: L. Prieto) antibodies and as loading control anti-β-actin, anti-GAPDH, or anti-β tubulin (Cell Signaling Technology) according to standard procedures.

    Techniques: Incubation, Mutagenesis, In Vitro, Recombinant, Immunoprecipitation, Expressing, Transfection, Isolation, Staining

    DENV infection activates cGAS-dependent antiviral responses. ( A ) In vitro assay for functional cGAS expression in A549 and THP-1 cell lysates as indicated by the production of cGAMP in the presence of 1 μg/ml poly(dA:dT). Results demonstrate that cGAS is functional in A549 as it is in THP-1, as shown previously 3 . ( B ) Western blot analysis of cGAS expression in A549 and THP-1 cells (control). THP-1 cells used as positive control and not as point of comparison for cGAS expression levels between A549 and THP-1 cell lines. ( C ) cGAMP production in A549 cells that are uninfected or infected with DENV2-PDK53 at MOI 1 for 24 hrs. Absorbance values normalized by cell count of each group. Results shown are representative of at least three independent experiments. ( D , E ) DENV RNA levels in infected A549 cells that were pre-treated ( D ) or post-treated ( E ) with 1 μg/ml cGAMP or 250 units/ml IFNα. Results are normalized by GAPDH expression and represent mean ± SD of at least four independent experiments. ( F ) DENV RNA levels in cGAS knockdown (si-cGAS) A549 cells compared to siRNA scrambled control (si-control) when infected with DENV2-PDK53 at MOI 1 for 24 hrs, assessed by RT-qPCR and normalized by GAPDH expression. Western blot confirmation of decreased cGAS expression in si-cGAS A549 cells, normalized with β-actin. Data is represented as mean ± SD of at least three independent experiments. In this figure, *depicts P

    Journal: Scientific Reports

    Article Title: Dengue virus activates cGAS through the release of mitochondrial DNA

    doi: 10.1038/s41598-017-03932-1

    Figure Lengend Snippet: DENV infection activates cGAS-dependent antiviral responses. ( A ) In vitro assay for functional cGAS expression in A549 and THP-1 cell lysates as indicated by the production of cGAMP in the presence of 1 μg/ml poly(dA:dT). Results demonstrate that cGAS is functional in A549 as it is in THP-1, as shown previously 3 . ( B ) Western blot analysis of cGAS expression in A549 and THP-1 cells (control). THP-1 cells used as positive control and not as point of comparison for cGAS expression levels between A549 and THP-1 cell lines. ( C ) cGAMP production in A549 cells that are uninfected or infected with DENV2-PDK53 at MOI 1 for 24 hrs. Absorbance values normalized by cell count of each group. Results shown are representative of at least three independent experiments. ( D , E ) DENV RNA levels in infected A549 cells that were pre-treated ( D ) or post-treated ( E ) with 1 μg/ml cGAMP or 250 units/ml IFNα. Results are normalized by GAPDH expression and represent mean ± SD of at least four independent experiments. ( F ) DENV RNA levels in cGAS knockdown (si-cGAS) A549 cells compared to siRNA scrambled control (si-control) when infected with DENV2-PDK53 at MOI 1 for 24 hrs, assessed by RT-qPCR and normalized by GAPDH expression. Western blot confirmation of decreased cGAS expression in si-cGAS A549 cells, normalized with β-actin. Data is represented as mean ± SD of at least three independent experiments. In this figure, *depicts P

    Article Snippet: Primary antibodies included human cGAS (Sigma-Aldrich HPA031700, 1:500) and human β-actin (Cell Signaling 8H10D10, 1:1000).

    Techniques: Infection, In Vitro, Functional Assay, Expressing, Western Blot, Positive Control, Cell Counting, Quantitative RT-PCR

    MiR-216b suppresses HeLa cell proliferation by inhibiting FOXM1. a The expression levels of FOXM1 (upper left) and miR-216b (bottom left) in cervical cancer tissues of 8 patients were determined by western blotting and real-time RT-PCR, respectively. The relative band intensity values of FOXM1 normalized by β-actin were shown above the β-actin bands. The levels of miR-216b and FOXM1 in cervical cancer tissues were negatively correlated. Correlation analysis (right) confirmed the negative correlation between miR-216b and FOXM1. b FOXM1 expression was suppressed by FOXM1-siRNA in both negative control HeLa cells (NC) and miR-216b inhibitors transfected cells (miR-216b-in). Normalized FOXM1 expression was shown. HeLa cells were transfected with or without FOXM1-siRNA and miR-216b inhibitors and measured by western blotting analysis. β-actin served as an internal control. * P

    Journal: BMC Cancer

    Article Title: MiR-216b inhibits cell proliferation by targeting FOXM1 in cervical cancer cells and is associated with better prognosis

    doi: 10.1186/s12885-017-3650-5

    Figure Lengend Snippet: MiR-216b suppresses HeLa cell proliferation by inhibiting FOXM1. a The expression levels of FOXM1 (upper left) and miR-216b (bottom left) in cervical cancer tissues of 8 patients were determined by western blotting and real-time RT-PCR, respectively. The relative band intensity values of FOXM1 normalized by β-actin were shown above the β-actin bands. The levels of miR-216b and FOXM1 in cervical cancer tissues were negatively correlated. Correlation analysis (right) confirmed the negative correlation between miR-216b and FOXM1. b FOXM1 expression was suppressed by FOXM1-siRNA in both negative control HeLa cells (NC) and miR-216b inhibitors transfected cells (miR-216b-in). Normalized FOXM1 expression was shown. HeLa cells were transfected with or without FOXM1-siRNA and miR-216b inhibitors and measured by western blotting analysis. β-actin served as an internal control. * P

    Article Snippet: Immonodetection was performed using antibodies including rabbit anti-FOXM1 polyclonal antibody, anti-cyclinD1, anti-p21, anti-LEF1, anti-c-myc, anti-Rb, anti- phosphorylated –Rb, and β-actin antibodies (Cell Signaling Technology, Danvers, MA, USA) at the dilution ratio of 1:1000.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Negative Control, Transfection

    MiR-216b decreases cyclinD1, c-myc, p-Rb and LEF1 level and enhances p21 expression. a Western blotting analysis showed that miR-216b mimics decreased the relative expression of cyclin D1, c-myc, LEF1 and phosphorylated Rb (p-Rb) but increased p21 level in HeLa cells 48 h after transfection, and miR-216b inhibitors had the opposite effect, as normalized by β-actin. * P

    Journal: BMC Cancer

    Article Title: MiR-216b inhibits cell proliferation by targeting FOXM1 in cervical cancer cells and is associated with better prognosis

    doi: 10.1186/s12885-017-3650-5

    Figure Lengend Snippet: MiR-216b decreases cyclinD1, c-myc, p-Rb and LEF1 level and enhances p21 expression. a Western blotting analysis showed that miR-216b mimics decreased the relative expression of cyclin D1, c-myc, LEF1 and phosphorylated Rb (p-Rb) but increased p21 level in HeLa cells 48 h after transfection, and miR-216b inhibitors had the opposite effect, as normalized by β-actin. * P

    Article Snippet: Immonodetection was performed using antibodies including rabbit anti-FOXM1 polyclonal antibody, anti-cyclinD1, anti-p21, anti-LEF1, anti-c-myc, anti-Rb, anti- phosphorylated –Rb, and β-actin antibodies (Cell Signaling Technology, Danvers, MA, USA) at the dilution ratio of 1:1000.

    Techniques: Expressing, Western Blot, Transfection

    Fluorescence activated cell sorting (FACS) analysis of stained cells and validation of cell cycle sorted populations in HDFa, NCI-H295R and HeLa cells. Panels a - c : FACS analysis shows cell cycle distribution of stained cells before (first image) and after sorting to G1, S and G2 phases (second, third and fourth images, respectively; Panel a : HDFa, Panel b : NCI-H295R, Panel c : HeLa. Intervals of fluorescence intensity (shown as vertical bands) were defined to gate G1, S and G2 phases, respectively. Panels d-e : Western blot analysis confirms the high efficacy of cell cycle sort (Panel d : NCI-H295R, Panel e : HeLa). Density values of phospho(Tyr15)-CDC-2 were first normalized to β-actin loading control and were further normalized to G1 phase (displayed above each band)

    Journal: BMC Genomics

    Article Title: Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression

    doi: 10.1186/s12864-016-2747-6

    Figure Lengend Snippet: Fluorescence activated cell sorting (FACS) analysis of stained cells and validation of cell cycle sorted populations in HDFa, NCI-H295R and HeLa cells. Panels a - c : FACS analysis shows cell cycle distribution of stained cells before (first image) and after sorting to G1, S and G2 phases (second, third and fourth images, respectively; Panel a : HDFa, Panel b : NCI-H295R, Panel c : HeLa. Intervals of fluorescence intensity (shown as vertical bands) were defined to gate G1, S and G2 phases, respectively. Panels d-e : Western blot analysis confirms the high efficacy of cell cycle sort (Panel d : NCI-H295R, Panel e : HeLa). Density values of phospho(Tyr15)-CDC-2 were first normalized to β-actin loading control and were further normalized to G1 phase (displayed above each band)

    Article Snippet: Thereafter membranes were stripped with mild stripping buffer (0.2 M glycine, 0.1 % sodium dodecyl sulfate, 0.1 % Tween-20, pH = 2.2) by gentle agitation for 45 min at room temperature, and were blocked again for subsequent detection of loading control β-actin (Cell Signaling Technology, cat. No.: 4967, dilution: 1:2000).

    Techniques: Fluorescence, FACS, Staining, Western Blot

    Regulation of ALDH1A1 expression by tamoxifen-activated ERα36. (A) Putative sites in ERα36 involved in interaction with 4-OHT. All aa residues close to 4-OHT in less than 4 Å are shown by lines. The analysis was performed with Discovery Studio 2.0 (Accelrys Software Inc.). (B) Binding of 4-OHT to purified GST-ERα36 fusion protein. GST-ERα36 was immobilized to an SPR sensor chip by GST capturing and 4-OHT was introduced as the soluble-phase analyte. The sensorgrams reached equilibrium and rapidly returned to baseline, demonstrating quick interaction kinetics between GST-ERα36 and 4-OHT. The KD was estimated as 11.6 ± 1.0 μM using the Biacore Evaluation Software. (C) Nuclear localization of ERα36 (green) in MDA-MB 436 cells after treatment with 4-OHT (1 μM) for 20 and 40 min. Heochst (blue) was used for nuclear staining. Scale bar, 20 μm. (D) Western blot of ERα36 in the cytoplasm or nuclei of MDA-MB 436-ALDH1 high cells after 4-OHT or E2 treatment. Lamin-B1 was used as a nuclear protein control, β-actin as a cytoplasm protein control. C, cytoplasm; N, nuclei. (E) HS578 ERE-luciferase assays showing the transcriptional ability of ERα36 activated by E2 or 4-OHT. ERα36 or ERα66 was transfected into HS578 cells along with an ERE-luciferase element. The transcriptional activity was measured. Error bars represent SEM from mean of triplicate samples. (F) Two potential ERE-binding sites in the ALDH1A1 promoter as analyzed by Transcription Element Search System. (G) ChIP/PCR analysis of MDA-MB-436 cell lysates showing endogenous ERα36 bound to ALDH1A1 promoter after treatment with E2 (1 nM) or 4-OHT (1 μM). An unrelated mouse IgG was used as an immunoprecipitation control. * P

    Journal: Cell Research

    Article Title: Tamoxifen enhances stemness and promotes metastasis of ERα36+ breast cancer by upregulating ALDH1A1 in cancer cells

    doi: 10.1038/cr.2018.15

    Figure Lengend Snippet: Regulation of ALDH1A1 expression by tamoxifen-activated ERα36. (A) Putative sites in ERα36 involved in interaction with 4-OHT. All aa residues close to 4-OHT in less than 4 Å are shown by lines. The analysis was performed with Discovery Studio 2.0 (Accelrys Software Inc.). (B) Binding of 4-OHT to purified GST-ERα36 fusion protein. GST-ERα36 was immobilized to an SPR sensor chip by GST capturing and 4-OHT was introduced as the soluble-phase analyte. The sensorgrams reached equilibrium and rapidly returned to baseline, demonstrating quick interaction kinetics between GST-ERα36 and 4-OHT. The KD was estimated as 11.6 ± 1.0 μM using the Biacore Evaluation Software. (C) Nuclear localization of ERα36 (green) in MDA-MB 436 cells after treatment with 4-OHT (1 μM) for 20 and 40 min. Heochst (blue) was used for nuclear staining. Scale bar, 20 μm. (D) Western blot of ERα36 in the cytoplasm or nuclei of MDA-MB 436-ALDH1 high cells after 4-OHT or E2 treatment. Lamin-B1 was used as a nuclear protein control, β-actin as a cytoplasm protein control. C, cytoplasm; N, nuclei. (E) HS578 ERE-luciferase assays showing the transcriptional ability of ERα36 activated by E2 or 4-OHT. ERα36 or ERα66 was transfected into HS578 cells along with an ERE-luciferase element. The transcriptional activity was measured. Error bars represent SEM from mean of triplicate samples. (F) Two potential ERE-binding sites in the ALDH1A1 promoter as analyzed by Transcription Element Search System. (G) ChIP/PCR analysis of MDA-MB-436 cell lysates showing endogenous ERα36 bound to ALDH1A1 promoter after treatment with E2 (1 nM) or 4-OHT (1 μM). An unrelated mouse IgG was used as an immunoprecipitation control. * P

    Article Snippet: Primary antibodies (ERα36, ERα66 and β-actin) were added overnight at 4 °C, followed by incubation with a secondary antibody at room temperature for 2 h. The antibody against β-actin as a control was from Cell Signaling Technology (#9559).

    Techniques: Expressing, Software, Binding Assay, Purification, SPR Assay, Chromatin Immunoprecipitation, Multiple Displacement Amplification, Staining, Western Blot, Luciferase, Transfection, Activity Assay, Polymerase Chain Reaction, Immunoprecipitation

    Endogenous ANXA1 inhibits InsP 3 R-mediated Ca 2+ release. ( A ) Western blot of ANXA1 and β-actin in lysates from HEKtsA201 cells treated with transfection medium (left) or ANXA1 siRNA (right) (one of four similar blots shown). ( B ) Summary of ANXA1 protein knockdown. HEKtsA201 cells treated with ANXA1 siRNA (four samples), transfection medium only (two samples) or with non-targeting (N-T) siRNA (two samples). ( C ) Typical trace of fura-2 fluorescence ratio (Δ R ) observed in population of intact HEKtsA201 cells stimulated by 10 μM carbachol (CCH). Maximal rise in Δ R (Δ R max ): blue double arrow; time constant ( τ ) of single exponential fit to rising phase of fluorescence ratio (red curve) is time for Δ R to rise to [1–1/ e ] of Δ R max : red double arrow. ( D ) Normalized Δ R max of individual Δ R traces (circles) and averages and s.e.m. (horizontal bars) for ANXA1 siRNA-treated (right) and control cells (left). Number of traces tabulated next to horizontal bars. ( E ) Normalized rate of change of Δ R (1/ τ ) for Δ R traces using convention as ( D ). ( F ) ANXA1 immunofluorescence intensity of HeLa cells treated with non-targeting (N-T) or ANXA1 siRNA. Number of cells tabulated below corresponding circles. ( G ) Fractions of N-T or ANXA1 siRNA-treated HeLa cells that responded by ER Ca 2+ release through InsP 3 R when stimulated by sub-saturating 10 (red) or saturating 100 (blue) μM histamine. ( H–I ) Traces of mean normalized ER Ca 2+ release from N-T (red) or ANXA1 (black) siRNA-treated HeLa cells responding to 100 μM ( H ) or 10 μM ( I ) histamine. ( J ) Fractions of N-T or ANXA1 siRNA-treated HeLa cells that oscillated in response to 10 (red) or 100 (blue) μM histamine. ( K ) Selected traces showing different kinds of Ca 2+ signals in AnxA1 siRNA-treated HeLa cells responding to sub-saturating 10 μM histamine. ( L ) Typical fluorescence amplitude (ΔF/F 0 ) traces showing local Ca 2+ release events (puffs) in HEK293 cells treated with N-T (black) or ANXA1 (red) siRNA. Cells stimulated by photolysis of caged i-InsP 3 using sub-maximal 50 ms UV flash. ( M–P ) Puffs generated by 50 ms UV flash were subsequently observed for 30 s in eight imaging fields ( M ); puffs generated by maximal 150 ms UV flash and subsequently observed for 10 s in six imaging fields ( N ). Dots indicate numbers of puffs observed for N-T (black) and ANXA1 (red) siRNA-treated cells. Means and s.e.m. indicated by bars. ( O–P ) Dot plots of ΔF/F 0 of individual Ca 2+ puffs observed in N-T (black) and ANXA1 (red) siRNA-treated cells, generated by 50 ms ( O ) and 150 ms ( P ) UV flashes, respectively. Means and s.e.m. indicated by diamonds and bars, respectively.

    Journal: eLife

    Article Title: ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein

    doi: 10.7554/eLife.53531

    Figure Lengend Snippet: Endogenous ANXA1 inhibits InsP 3 R-mediated Ca 2+ release. ( A ) Western blot of ANXA1 and β-actin in lysates from HEKtsA201 cells treated with transfection medium (left) or ANXA1 siRNA (right) (one of four similar blots shown). ( B ) Summary of ANXA1 protein knockdown. HEKtsA201 cells treated with ANXA1 siRNA (four samples), transfection medium only (two samples) or with non-targeting (N-T) siRNA (two samples). ( C ) Typical trace of fura-2 fluorescence ratio (Δ R ) observed in population of intact HEKtsA201 cells stimulated by 10 μM carbachol (CCH). Maximal rise in Δ R (Δ R max ): blue double arrow; time constant ( τ ) of single exponential fit to rising phase of fluorescence ratio (red curve) is time for Δ R to rise to [1–1/ e ] of Δ R max : red double arrow. ( D ) Normalized Δ R max of individual Δ R traces (circles) and averages and s.e.m. (horizontal bars) for ANXA1 siRNA-treated (right) and control cells (left). Number of traces tabulated next to horizontal bars. ( E ) Normalized rate of change of Δ R (1/ τ ) for Δ R traces using convention as ( D ). ( F ) ANXA1 immunofluorescence intensity of HeLa cells treated with non-targeting (N-T) or ANXA1 siRNA. Number of cells tabulated below corresponding circles. ( G ) Fractions of N-T or ANXA1 siRNA-treated HeLa cells that responded by ER Ca 2+ release through InsP 3 R when stimulated by sub-saturating 10 (red) or saturating 100 (blue) μM histamine. ( H–I ) Traces of mean normalized ER Ca 2+ release from N-T (red) or ANXA1 (black) siRNA-treated HeLa cells responding to 100 μM ( H ) or 10 μM ( I ) histamine. ( J ) Fractions of N-T or ANXA1 siRNA-treated HeLa cells that oscillated in response to 10 (red) or 100 (blue) μM histamine. ( K ) Selected traces showing different kinds of Ca 2+ signals in AnxA1 siRNA-treated HeLa cells responding to sub-saturating 10 μM histamine. ( L ) Typical fluorescence amplitude (ΔF/F 0 ) traces showing local Ca 2+ release events (puffs) in HEK293 cells treated with N-T (black) or ANXA1 (red) siRNA. Cells stimulated by photolysis of caged i-InsP 3 using sub-maximal 50 ms UV flash. ( M–P ) Puffs generated by 50 ms UV flash were subsequently observed for 30 s in eight imaging fields ( M ); puffs generated by maximal 150 ms UV flash and subsequently observed for 10 s in six imaging fields ( N ). Dots indicate numbers of puffs observed for N-T (black) and ANXA1 (red) siRNA-treated cells. Means and s.e.m. indicated by bars. ( O–P ) Dot plots of ΔF/F 0 of individual Ca 2+ puffs observed in N-T (black) and ANXA1 (red) siRNA-treated cells, generated by 50 ms ( O ) and 150 ms ( P ) UV flashes, respectively. Means and s.e.m. indicated by diamonds and bars, respectively.

    Article Snippet: Expression levels of ANXA1 in HEKtsA201 cells were probed in Western blots using anti-ANXA1 antibody (Proteintech cat. # 21990–1-AP, 1:1000 dilution), stripped and re-probed with anti-β-actin antibody (Cell Signalling cat. # 8H10D10, 1:1000 dilution).

    Techniques: Western Blot, Transfection, Fluorescence, Immunofluorescence, Generated, Imaging

    Expression of other IFT proteins in conditional Ift140 KO mice A. Representative Western blotting images showing testicular expression of IFT20, IFT25, IFT27, IFT74, IFT81, and IFT88 using specific antibodies in three control and three conditional Ift140 mice. β-actin was used as a loading control. B. Statistical analysis of relative expression of the IFT proteins normalized by β-actin. There is no difference in expression levels of these selective IFT proteins between the controls and the Ift140 KO mice. C. Localization of IFT20 in the germ cells of control and conditional Ift140 knockout mice. In mice with both genotypes, IFT20 is present in the Golgi bodies of spermatocytes (upper panels), acrosome of round spermatids (middle panels), and manchette of elongating spermatids (lower panels). However, in the control animal, IFT20 appears to attach closely to the nuclear membrane (white arrow) in round spermatids; in the Ift140 KO, IFT20 does not seem to attach tightly to the nuclear membrane (dashed white arrow). D. Abnormal localization of IFT27 in the conditional Ift140 knockout mice. The granule-like pattern of the IFT27 signal was never observed in the control mice. E. Abnormal localization of IFT88 in the conditional Ift140 knockout mice. In the control mouse, IFT88 is present in the cytoplasm of spermatocytes and round spermatids, manchette of elongating spermatids, and the developing tails; In the Ift140 KO mice, even though IFT88 is still present in the cytoplasm of spermatocytes and round spermatids, it accumulates abnormally in the germ cells at later stages (white arrow heads).

    Journal: Cytoskeleton (Hoboken, N.J.)

    Article Title: Intraflagellar Transporter Protein 140 (IFT140), a component of IFT-A complex, is Essential for Male Fertility and Spermiogenesis in Mice

    doi: 10.1002/cm.21427

    Figure Lengend Snippet: Expression of other IFT proteins in conditional Ift140 KO mice A. Representative Western blotting images showing testicular expression of IFT20, IFT25, IFT27, IFT74, IFT81, and IFT88 using specific antibodies in three control and three conditional Ift140 mice. β-actin was used as a loading control. B. Statistical analysis of relative expression of the IFT proteins normalized by β-actin. There is no difference in expression levels of these selective IFT proteins between the controls and the Ift140 KO mice. C. Localization of IFT20 in the germ cells of control and conditional Ift140 knockout mice. In mice with both genotypes, IFT20 is present in the Golgi bodies of spermatocytes (upper panels), acrosome of round spermatids (middle panels), and manchette of elongating spermatids (lower panels). However, in the control animal, IFT20 appears to attach closely to the nuclear membrane (white arrow) in round spermatids; in the Ift140 KO, IFT20 does not seem to attach tightly to the nuclear membrane (dashed white arrow). D. Abnormal localization of IFT27 in the conditional Ift140 knockout mice. The granule-like pattern of the IFT27 signal was never observed in the control mice. E. Abnormal localization of IFT88 in the conditional Ift140 knockout mice. In the control mouse, IFT88 is present in the cytoplasm of spermatocytes and round spermatids, manchette of elongating spermatids, and the developing tails; In the Ift140 KO mice, even though IFT88 is still present in the cytoplasm of spermatocytes and round spermatids, it accumulates abnormally in the germ cells at later stages (white arrow heads).

    Article Snippet: The membranes were immunoblotted with indicated antibodies at 4°C overnight: anti-IFT140, anti-IFT88, anti-IFT20 and anti-IFT27 (1:2000, Dr. Pazour’s laboratory); anti-IFT25 (1:2000, Protein Tech, 15732-1-AP); anti-IFT74 (1:1000, Antibody Verify); anti-IFT81 (1:1000, Proteintech Group); anti-β-actin (1:2000, Cell Signaling).

    Techniques: Expressing, Mouse Assay, Western Blot, Knock-Out

    HBxΔ127 has a greater capacity to stimulate SREBP-1c relative to wild type HBx. (A, B) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were transfected with pSilencer 3.0-X plasmid for 48 h. (A) The mRNA levels of SREBP-1c were detected by RT-PCR. (B) Immunoblot analysis to detect SREBP-1c (top panel) and β-actin protein levels (bottom panel). (C, D) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were co-transfected with or without pSilencer 3.0-X plasmid and SREBP-1c-571-Luc-WT. Data are representative of 3 independent experiments. Values represent means±SD. n =3. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: A mutant of HBx (HBxΔ127) promotes hepatoma cell growth via sterol regulatory element binding protein 1c involving 5-lipoxygenase

    doi: 10.1038/aps.2010.5

    Figure Lengend Snippet: HBxΔ127 has a greater capacity to stimulate SREBP-1c relative to wild type HBx. (A, B) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were transfected with pSilencer 3.0-X plasmid for 48 h. (A) The mRNA levels of SREBP-1c were detected by RT-PCR. (B) Immunoblot analysis to detect SREBP-1c (top panel) and β-actin protein levels (bottom panel). (C, D) HepG2-X (or H7402-X) cells and HepG2-XΔ127 (or H7402-XΔ127) cells were co-transfected with or without pSilencer 3.0-X plasmid and SREBP-1c-571-Luc-WT. Data are representative of 3 independent experiments. Values represent means±SD. n =3. b P

    Article Snippet: For protein loading controls, the amount of β-actin protein was also determined using a β-actin-specific antibody (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction