glyceraldehyde 3 phosphate dehydrogenase gapdh  (Millipore)


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    Structured Review

    Millipore glyceraldehyde 3 phosphate dehydrogenase gapdh
    Androgen receptor (AR) + triple-negative breast cancer (TNBC) cell lines have activated PI3K signaling and respond to dihydrotestosterone (DHT) stimulation (A) Graphs display relative viability of the prostate cancer cell line (LNCAP, red line) compared to each of the AR + TNBC cell lines (blue line) 5 days after addition of increasing doses of DHT in charcoal-stripped media. (B) Heatmap displays relative protein levels (reverse-phase protein array, RPPA) of AR, p-AKT (S473 and T308), p-GSK3β (S9 and S21) and PTEN across indicated TNBC cell lines. Unsupervised hierarchical clustering was performed on PI3K pathway proteins (PTEN, p-AKT and p-GSK3β). Known PI3K pathway aberrations are indicated in the colorbar as PTEN loss (blue) or PIK3CA mutations (red). (C) Immunoblot displays relative protein levels of AR, p-AKT, p-S6 in AR-expressing prostate cancer (LNCaP), primary cultures of human mammary epithelial cells (HMECs) and the indicated LAR TNBC cell lines. <t>Glyceraldehyde-3-phosphate</t> dehydrogenase <t>(GAPDH)</t> serves as a loading control. (D) Immunohistochemistry of indicated proteins was performed on cell lines to assess levels of AR and p-AKT (S473). Results are representative of three independent experiments.
    Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors"

    Article Title: PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-014-0406-x

    Androgen receptor (AR) + triple-negative breast cancer (TNBC) cell lines have activated PI3K signaling and respond to dihydrotestosterone (DHT) stimulation (A) Graphs display relative viability of the prostate cancer cell line (LNCAP, red line) compared to each of the AR + TNBC cell lines (blue line) 5 days after addition of increasing doses of DHT in charcoal-stripped media. (B) Heatmap displays relative protein levels (reverse-phase protein array, RPPA) of AR, p-AKT (S473 and T308), p-GSK3β (S9 and S21) and PTEN across indicated TNBC cell lines. Unsupervised hierarchical clustering was performed on PI3K pathway proteins (PTEN, p-AKT and p-GSK3β). Known PI3K pathway aberrations are indicated in the colorbar as PTEN loss (blue) or PIK3CA mutations (red). (C) Immunoblot displays relative protein levels of AR, p-AKT, p-S6 in AR-expressing prostate cancer (LNCaP), primary cultures of human mammary epithelial cells (HMECs) and the indicated LAR TNBC cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a loading control. (D) Immunohistochemistry of indicated proteins was performed on cell lines to assess levels of AR and p-AKT (S473). Results are representative of three independent experiments.
    Figure Legend Snippet: Androgen receptor (AR) + triple-negative breast cancer (TNBC) cell lines have activated PI3K signaling and respond to dihydrotestosterone (DHT) stimulation (A) Graphs display relative viability of the prostate cancer cell line (LNCAP, red line) compared to each of the AR + TNBC cell lines (blue line) 5 days after addition of increasing doses of DHT in charcoal-stripped media. (B) Heatmap displays relative protein levels (reverse-phase protein array, RPPA) of AR, p-AKT (S473 and T308), p-GSK3β (S9 and S21) and PTEN across indicated TNBC cell lines. Unsupervised hierarchical clustering was performed on PI3K pathway proteins (PTEN, p-AKT and p-GSK3β). Known PI3K pathway aberrations are indicated in the colorbar as PTEN loss (blue) or PIK3CA mutations (red). (C) Immunoblot displays relative protein levels of AR, p-AKT, p-S6 in AR-expressing prostate cancer (LNCaP), primary cultures of human mammary epithelial cells (HMECs) and the indicated LAR TNBC cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a loading control. (D) Immunohistochemistry of indicated proteins was performed on cell lines to assess levels of AR and p-AKT (S473). Results are representative of three independent experiments.

    Techniques Used: Protein Array, Expressing, Immunohistochemistry

    Pharmacological targeting of androgen receptor (AR) with bicalutamide (CDX) is additive in combination with of GDC0941 and GDC0980 in AR + triple-negative breast cancer (TNBC) cell lines. (A) Line graphs show viability of AR + cell lines treated with increasing concentrations of GDC-0941 (top) or GDC-0980 (bottom) alone (blue) or in combination (red) with 25 μM CDX. Dashed black line depicts the theoretical line of additivity of both drugs determined from the effect of CDX alone and either GDC-0941 or GDC-0980 alone. Error bars represent SD for three independent experiments. (B) Immunoblots from AR + TNBC cell lines treated with either CDX (25 μM), GDC-0941 (300 nM) or GDC0980 (100 nM) as single agents or CDX in combination with either GDC-0941 or GDC-0980 for 48 h analyzed for AR, p-AKT, AKT, p-S6, S6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein.
    Figure Legend Snippet: Pharmacological targeting of androgen receptor (AR) with bicalutamide (CDX) is additive in combination with of GDC0941 and GDC0980 in AR + triple-negative breast cancer (TNBC) cell lines. (A) Line graphs show viability of AR + cell lines treated with increasing concentrations of GDC-0941 (top) or GDC-0980 (bottom) alone (blue) or in combination (red) with 25 μM CDX. Dashed black line depicts the theoretical line of additivity of both drugs determined from the effect of CDX alone and either GDC-0941 or GDC-0980 alone. Error bars represent SD for three independent experiments. (B) Immunoblots from AR + TNBC cell lines treated with either CDX (25 μM), GDC-0941 (300 nM) or GDC0980 (100 nM) as single agents or CDX in combination with either GDC-0941 or GDC-0980 for 48 h analyzed for AR, p-AKT, AKT, p-S6, S6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein.

    Techniques Used: Western Blot

    2) Product Images from "Homocysteine Increases Tau Phosphorylation, Truncation and Oligomerization"

    Article Title: Homocysteine Increases Tau Phosphorylation, Truncation and Oligomerization

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19030891

    Total tau protein was increased by L-homocysteine (L-Hcy) in M1C cells. M1C cells were subjected to 5 days of tau expression (by reducing tetracycline) and cells were exposed to 10, 100, 1000 µM L-Hcy during the final 24 h of tau induction. The amount of Tau5 positive tau was increased in a dose-dependent manner when cells were treated with L-Hcy. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Bar: ± SD, ** p
    Figure Legend Snippet: Total tau protein was increased by L-homocysteine (L-Hcy) in M1C cells. M1C cells were subjected to 5 days of tau expression (by reducing tetracycline) and cells were exposed to 10, 100, 1000 µM L-Hcy during the final 24 h of tau induction. The amount of Tau5 positive tau was increased in a dose-dependent manner when cells were treated with L-Hcy. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Bar: ± SD, ** p

    Techniques Used: Expressing

    3) Product Images from "Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation"

    Article Title: Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0754-9

    Metformin inhibited morphine-induced inflammation in microglial BV-2 cells. Cells were pretreated with metformin (4, 20, or 100 μM) for 15 min before the challenge of morphine (200 μM). BV-2 cells were collected and analyzed 6 h after morphine was given. a – c Metformin suppressed the expression of pro-inflammatory factors induced by morphine in BV-2 cells in a dose-dependent manner ( n = 4). The levels of IL-1β, IL-6, and TNF-α mRNA were determined using real-time quantitative polymerase chain reaction (PCR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an invariant control. d Metformin inhibited the phosphorylation of p38 induced by morphine treatment in BV-2 cells in a dose-dependent manner ( n = 4). e Metformin suppressed TLR-4 mRNA expression induced by morphine in BV-2 cells in a dose-dependent manner ( n = 4). The level of TLR-4 mRNA was determined with real-time quantitative PCR. (* p
    Figure Legend Snippet: Metformin inhibited morphine-induced inflammation in microglial BV-2 cells. Cells were pretreated with metformin (4, 20, or 100 μM) for 15 min before the challenge of morphine (200 μM). BV-2 cells were collected and analyzed 6 h after morphine was given. a – c Metformin suppressed the expression of pro-inflammatory factors induced by morphine in BV-2 cells in a dose-dependent manner ( n = 4). The levels of IL-1β, IL-6, and TNF-α mRNA were determined using real-time quantitative polymerase chain reaction (PCR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an invariant control. d Metformin inhibited the phosphorylation of p38 induced by morphine treatment in BV-2 cells in a dose-dependent manner ( n = 4). e Metformin suppressed TLR-4 mRNA expression induced by morphine in BV-2 cells in a dose-dependent manner ( n = 4). The level of TLR-4 mRNA was determined with real-time quantitative PCR. (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    4) Product Images from "Potential Role of Thymosin-α1 Adjuvant Therapy for Glioblastoma"

    Article Title: Potential Role of Thymosin-α1 Adjuvant Therapy for Glioblastoma

    Journal: Journal of Oncology

    doi: 10.1155/2009/302084

    Increased proapoptosis gene products in Talpha1-treated 9L glioblastoma cells. 9L cells seeded in 96-well plates were treated with 10 mM Talpha1 for 24 hours. After fixation and permeabilization, immunoreactivity was measured directly in the cells using the microtiter immunocytochemical ELISA (MICE) assay. Levels of immunoreactivity were normalized to cell density and the corresponding ratio represents the MICE index. The graph depicts the mean ± S.D. of the levels of proliferating cell nuclear antigen (PCNA), Bcl-2, p21, p53, Fas L, Fas R, tumor necrosis factor receptor, type 1 (TNF R1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) measured in 8 replicate vehicle-treated or Talpha1-treated cultures. Asterisks denote significant differences from control ( P
    Figure Legend Snippet: Increased proapoptosis gene products in Talpha1-treated 9L glioblastoma cells. 9L cells seeded in 96-well plates were treated with 10 mM Talpha1 for 24 hours. After fixation and permeabilization, immunoreactivity was measured directly in the cells using the microtiter immunocytochemical ELISA (MICE) assay. Levels of immunoreactivity were normalized to cell density and the corresponding ratio represents the MICE index. The graph depicts the mean ± S.D. of the levels of proliferating cell nuclear antigen (PCNA), Bcl-2, p21, p53, Fas L, Fas R, tumor necrosis factor receptor, type 1 (TNF R1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) measured in 8 replicate vehicle-treated or Talpha1-treated cultures. Asterisks denote significant differences from control ( P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    5) Product Images from "Anti-TNF certolizumab pegol induces antioxidant response in human monocytes via reverse signaling"

    Article Title: Anti-TNF certolizumab pegol induces antioxidant response in human monocytes via reverse signaling

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-016-0955-8

    Heme oxygenase 1 ( HO-1 ) induction by anti-TNF certolizumab pegol ( CZP ) is due to reverse signaling. Monocytes were incubated, or not, with CZP (5 μg/ml) for 16 h in the presence of TNF receptor 1 ( TNFR1 ) and TNFR2 (5 μg/ml) blocking antibody. HO-1 protein expression was assessed by western blot ( a ). Quantification of four western blot experiments was performed. Paired t test p = 0.05 ( b ). GAPDH glyceraldehyde 3-phosphate dehydrogenase, Abs antibodies
    Figure Legend Snippet: Heme oxygenase 1 ( HO-1 ) induction by anti-TNF certolizumab pegol ( CZP ) is due to reverse signaling. Monocytes were incubated, or not, with CZP (5 μg/ml) for 16 h in the presence of TNF receptor 1 ( TNFR1 ) and TNFR2 (5 μg/ml) blocking antibody. HO-1 protein expression was assessed by western blot ( a ). Quantification of four western blot experiments was performed. Paired t test p = 0.05 ( b ). GAPDH glyceraldehyde 3-phosphate dehydrogenase, Abs antibodies

    Techniques Used: Incubation, Blocking Assay, Expressing, Western Blot

    6) Product Images from "Identification and isolation of kidney-derived stem cells from transgenic rats with diphtheria toxin-induced kidney damage"

    Article Title: Identification and isolation of kidney-derived stem cells from transgenic rats with diphtheria toxin-induced kidney damage

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2016.3516

    Reverse transcription-polymerase chain reaction analysis. Expression profile of (A) Pax-2, (B) Oct-4 and (C) GAPDH in the isolated kidney-derived stem cells. The reactions were run in triplicate. PAX-2, paired box 2; Oct-4, octamer-binding transcription factor 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
    Figure Legend Snippet: Reverse transcription-polymerase chain reaction analysis. Expression profile of (A) Pax-2, (B) Oct-4 and (C) GAPDH in the isolated kidney-derived stem cells. The reactions were run in triplicate. PAX-2, paired box 2; Oct-4, octamer-binding transcription factor 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Derivative Assay, Binding Assay

    7) Product Images from "Imidazole Antifungal Drugs Inhibit the Cell Proliferation and Invasion of Human Breast Cancer Cells"

    Article Title: Imidazole Antifungal Drugs Inhibit the Cell Proliferation and Invasion of Human Breast Cancer Cells

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2018.042

    Effect of azole antifungal compounds on protein expression. MCF-7 and MDA-MB-231 cells were treated with 50 μM imidazole [clotrimazole (CTZ) or ketoconazole (KCZ)] or triazole [fluconazole (FCZ) or itraconazole (ICZ)] compounds for 24 h. The protein expression related to apoptosis (A) and cell cycle (B) were examined in MCF-7 and MDA-MB-231 cells after treatment of each compound for 24 h. The protein expression was also examined in the xenograft model of MDA-MB-231 cells after treatment of each compound for 8 weeks (C). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. This experiment was performed three times. Band density was estimated using ImageJ 1.45s software (NIH).
    Figure Legend Snippet: Effect of azole antifungal compounds on protein expression. MCF-7 and MDA-MB-231 cells were treated with 50 μM imidazole [clotrimazole (CTZ) or ketoconazole (KCZ)] or triazole [fluconazole (FCZ) or itraconazole (ICZ)] compounds for 24 h. The protein expression related to apoptosis (A) and cell cycle (B) were examined in MCF-7 and MDA-MB-231 cells after treatment of each compound for 24 h. The protein expression was also examined in the xenograft model of MDA-MB-231 cells after treatment of each compound for 8 weeks (C). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. This experiment was performed three times. Band density was estimated using ImageJ 1.45s software (NIH).

    Techniques Used: Expressing, Multiple Displacement Amplification, Software

    8) Product Images from "Effects of Progesterone Treatment on Expression of Genes Involved in Uterine Quiescence"

    Article Title: Effects of Progesterone Treatment on Expression of Genes Involved in Uterine Quiescence

    Journal: Reproductive Sciences

    doi: 10.1177/1933719111398150

    Immuoblot of Maxi-K channel α-subunit (KCNMA1) in triplicate samples of cells treated either with estrogen (E) or estrogen plus progesterone (E + P). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) staining illustrates the uniformity of gel loading
    Figure Legend Snippet: Immuoblot of Maxi-K channel α-subunit (KCNMA1) in triplicate samples of cells treated either with estrogen (E) or estrogen plus progesterone (E + P). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) staining illustrates the uniformity of gel loading

    Techniques Used: Staining

    9) Product Images from "Glucocorticoid-induced leucine zipper (GILZ) is involved in glucocorticoid-induced and mineralocorticoid-induced leptin production by osteoarthritis synovial fibroblasts"

    Article Title: Glucocorticoid-induced leucine zipper (GILZ) is involved in glucocorticoid-induced and mineralocorticoid-induced leptin production by osteoarthritis synovial fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-016-1119-6

    Glucocorticoid-induced leucine zipper (GILZ) silencing did not alter prednisolone-induced glucocorticoid receptor ( GR ) degradation. Human osteoarthritis (OA) synovial fibroblasts were infected with a lentivirus expressing GILZ short hairpin RNA ( shRNA ) or with a control lentivirus. After 72 h, the cells were stimulated for 1, 4, 6, or 12 h with a glucocorticoid (1 μM prednisolone). Leptin expression was measured in the cell culture supernatants by ELISA. GILZ and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts was analyzed by western blotting. Upper panel , quantification results of western blot shown in the lower panel . Protein levels were normalized to GAPDH. Graph represents mean +/- SD (n = 3) patients. kD kiloDalton
    Figure Legend Snippet: Glucocorticoid-induced leucine zipper (GILZ) silencing did not alter prednisolone-induced glucocorticoid receptor ( GR ) degradation. Human osteoarthritis (OA) synovial fibroblasts were infected with a lentivirus expressing GILZ short hairpin RNA ( shRNA ) or with a control lentivirus. After 72 h, the cells were stimulated for 1, 4, 6, or 12 h with a glucocorticoid (1 μM prednisolone). Leptin expression was measured in the cell culture supernatants by ELISA. GILZ and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts was analyzed by western blotting. Upper panel , quantification results of western blot shown in the lower panel . Protein levels were normalized to GAPDH. Graph represents mean +/- SD (n = 3) patients. kD kiloDalton

    Techniques Used: Infection, Expressing, shRNA, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

    Transforming growth factor-β ( TGF-β ) decreased both prednisolone-induced leptin secretion and glucocorticoid-induced leucine zipper ( GILZ ) expression; Compound A ( CpdA ) did not induce GILZ expression. Human osteoarthritis (OA) synovial fibroblasts were stimulated for 5 days with prednisolone, TGF-β ( A ), or CpdA ( B ). Leptin expression was measured in the cell culture supernatants by ELISA. GILZ and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts was analyzed by western blotting. Right panels , quantification results of western blots shown in the left panels . Protein levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 3 patients). b *Significantly different from a ; c *significantly different from b ; e*significantly different from d ; f not significantly different from d
    Figure Legend Snippet: Transforming growth factor-β ( TGF-β ) decreased both prednisolone-induced leptin secretion and glucocorticoid-induced leucine zipper ( GILZ ) expression; Compound A ( CpdA ) did not induce GILZ expression. Human osteoarthritis (OA) synovial fibroblasts were stimulated for 5 days with prednisolone, TGF-β ( A ), or CpdA ( B ). Leptin expression was measured in the cell culture supernatants by ELISA. GILZ and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts was analyzed by western blotting. Right panels , quantification results of western blots shown in the left panels . Protein levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 3 patients). b *Significantly different from a ; c *significantly different from b ; e*significantly different from d ; f not significantly different from d

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

    Glucocorticoid-induced leucine zipper ( GILZ ) silencing inhibited glucocorticoid-induced and mineralocorticoid-induced leptin and leptin receptor ( Ob-R ) expression. Human osteoarthritis (OA) synovial fibroblasts were infected with three different lentiviruses expressing GILZ short hairpin RNA ( shRNA ) or with a control lentivirus (multiplicity of infection (MOI) 30 for a and b ; MOI 5, 10 and 20 for c ). After 72 h, the cells were stimulated for 5 days with a glucocorticoid (1 μM prednisolone) or mineralocorticoid (10 μM aldosterone). Leptin expression was measured in the cell culture supernatants by ELISA. Ob-R, GILZ and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts was analyzed by western blotting. Right panels , quantification results of western blots shown in the left panels . Protein levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 5 patients). b *Significantly different from a ; c *significantly different from b. kD kiloDalton
    Figure Legend Snippet: Glucocorticoid-induced leucine zipper ( GILZ ) silencing inhibited glucocorticoid-induced and mineralocorticoid-induced leptin and leptin receptor ( Ob-R ) expression. Human osteoarthritis (OA) synovial fibroblasts were infected with three different lentiviruses expressing GILZ short hairpin RNA ( shRNA ) or with a control lentivirus (multiplicity of infection (MOI) 30 for a and b ; MOI 5, 10 and 20 for c ). After 72 h, the cells were stimulated for 5 days with a glucocorticoid (1 μM prednisolone) or mineralocorticoid (10 μM aldosterone). Leptin expression was measured in the cell culture supernatants by ELISA. Ob-R, GILZ and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts was analyzed by western blotting. Right panels , quantification results of western blots shown in the left panels . Protein levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 5 patients). b *Significantly different from a ; c *significantly different from b. kD kiloDalton

    Techniques Used: Expressing, Infection, shRNA, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

    Glucocorticoid-induced leucine zipper ( GILZ ) expression is induced by prednisolone and aldosterone through glucocorticoid receptor ( GR ). a Human osteoarthritis (OA) synovial fibroblasts were pre-incubated or not for 1 h with a GR inhibitor (mifepristone) or mineralocorticoid receptor ( MR ) inhibitors (eplerenone and spironolactone) and were then stimulated for 5 days with a glucocorticoid (prednisolone) or a mineralocorticoid (aldosterone). GILZ and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts were analyzed by western blotting. Right panels , quantification results of western blots shown in left panels . Protein levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 4 patients). Significance was set at p
    Figure Legend Snippet: Glucocorticoid-induced leucine zipper ( GILZ ) expression is induced by prednisolone and aldosterone through glucocorticoid receptor ( GR ). a Human osteoarthritis (OA) synovial fibroblasts were pre-incubated or not for 1 h with a GR inhibitor (mifepristone) or mineralocorticoid receptor ( MR ) inhibitors (eplerenone and spironolactone) and were then stimulated for 5 days with a glucocorticoid (prednisolone) or a mineralocorticoid (aldosterone). GILZ and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts were analyzed by western blotting. Right panels , quantification results of western blots shown in left panels . Protein levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 4 patients). Significance was set at p

    Techniques Used: Expressing, Incubation, Western Blot

    Leptin secretion and leptin receptor ( Ob-R ) expression were induced by prednisolone and aldosterone through glucocorticoid receptor (GR) signaling. Human osteoarthritis (OA) synovial fibroblasts were pre-incubated or not for 1 h with a GR inhibitor (mifepristone) or mineralocorticoid receptor ( MR) inhibitors (eplerenone and spironolactone) and were then stimulated for 5 days with a glucocorticoid (prednisolone) ( A ) or mineralocorticoid (aldosterone) ( B , C ). Leptin expression was measured in cell culture supernatants by ELISA. Ob-R and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts were analyzed by western blotting. Upper panels ( right ), quantification results of western blots shown in bottom panels . Proteins levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 3 or 4 patients). Significance set at p
    Figure Legend Snippet: Leptin secretion and leptin receptor ( Ob-R ) expression were induced by prednisolone and aldosterone through glucocorticoid receptor (GR) signaling. Human osteoarthritis (OA) synovial fibroblasts were pre-incubated or not for 1 h with a GR inhibitor (mifepristone) or mineralocorticoid receptor ( MR) inhibitors (eplerenone and spironolactone) and were then stimulated for 5 days with a glucocorticoid (prednisolone) ( A ) or mineralocorticoid (aldosterone) ( B , C ). Leptin expression was measured in cell culture supernatants by ELISA. Ob-R and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression in whole-cell extracts were analyzed by western blotting. Upper panels ( right ), quantification results of western blots shown in bottom panels . Proteins levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 3 or 4 patients). Significance set at p

    Techniques Used: Expressing, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

    Leptin secretion and leptin receptor ( Ob-R ) expression were induced by prednisolone and aldosterone through glucocorticoid receptor (GR) signaling. Synovial fibroblasts were infected with three different lentiviruses expressing GR short hairpin RNA ( shRNA ) or with a non-target control lentivirus and were stimulated for 5 days with prednisolone or aldosterone. The GR inhibitor mifepristone was used as a positive control for GR inhibition. Leptin expression was measured in the cell culture supernatants by ELISA. Ob-R and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression were analyzed in whole-cell extracts by western blotting. Middle panel , quantification results of western blots shown in the lower panel . Protein levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 3 patients). Significance was set at p
    Figure Legend Snippet: Leptin secretion and leptin receptor ( Ob-R ) expression were induced by prednisolone and aldosterone through glucocorticoid receptor (GR) signaling. Synovial fibroblasts were infected with three different lentiviruses expressing GR short hairpin RNA ( shRNA ) or with a non-target control lentivirus and were stimulated for 5 days with prednisolone or aldosterone. The GR inhibitor mifepristone was used as a positive control for GR inhibition. Leptin expression was measured in the cell culture supernatants by ELISA. Ob-R and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression were analyzed in whole-cell extracts by western blotting. Middle panel , quantification results of western blots shown in the lower panel . Protein levels were normalized to GAPDH. Graphs represent mean +/- SD (n = 3 patients). Significance was set at p

    Techniques Used: Expressing, Infection, shRNA, Positive Control, Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

    10) Product Images from "Neonatal Androgenization Exacerbates Alcohol-Induced Liver Injury in Adult Rats, an Effect Abrogated by Estrogen"

    Article Title: Neonatal Androgenization Exacerbates Alcohol-Induced Liver Injury in Adult Rats, an Effect Abrogated by Estrogen

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029463

    Effects of ethanol and androgenization on profibrotic gene expression. Quantitative Real-Time PCR analysis of Interleukin-6 (IL-6) (black bar) and collagen Type 3 (COL3α1) (striped bar) gene expression in control (Ctrl), androgenized (Andro) and Andro + estrogen (Andro + E2) animals maintained on control Lieber-DeCarli (C-LDC) or ethanol-LDC (E-LDC) diet. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are presented as mean values ± SEM. *P
    Figure Legend Snippet: Effects of ethanol and androgenization on profibrotic gene expression. Quantitative Real-Time PCR analysis of Interleukin-6 (IL-6) (black bar) and collagen Type 3 (COL3α1) (striped bar) gene expression in control (Ctrl), androgenized (Andro) and Andro + estrogen (Andro + E2) animals maintained on control Lieber-DeCarli (C-LDC) or ethanol-LDC (E-LDC) diet. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are presented as mean values ± SEM. *P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    11) Product Images from "Inhibition of DYRK1A-EGFR axis by p53-MDM2 cascade mediates the induction of cellular senescence"

    Article Title: Inhibition of DYRK1A-EGFR axis by p53-MDM2 cascade mediates the induction of cellular senescence

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1521-5

    Negative regulation of DYRK1A by p53 is mediated by MDM2. a Downregulation of dual-specificity tyrosine-phosphorylated and tyrosine-regulated kinase 1A (DYRK1A) by p53 activation is attenuated by MG132. U2OS and A2780 cells were treated with 8 μM Nut3a for 48 h, and were then treated with 10 μM MG132 for the last 6 h before harvest. Protein levels of p53, epidermal growth factor receptor (EGFR), and DYRK1A were analyzed by western blot. b Nut3a shortens the half-life of DYRK1A proteins. U2OS cells were treated with dimethyl sulfoxide (DMSO) or 8 μM Nut3a for 17 h, and were then treated with 50 μg/mL cycloheximide for the indicated durations before harvest. Protein levels of DYRK1A were analyzed by western blot. Bottom: Signals on the immunoblots were analyzed by the ImageJ software (NIH, Bethesda, MD, USA) 45 and the DYRK1A protein amounts were normalized with that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). c Ectopic expression of MDM2 downregulates DYRK1A. pCMV-myc3-HDM2 (WT) and MDM2 C464A expression vectors were respectively transfected into U2OS cells using Lipofectamine 2000, and cells transfected with empty vector were as control. After 48 h, the cells extracts were examined by western blot for the determination of MDM2, DYRK1A. d MDM2 shortens the half-life of DYRK1A proteins. Co-transfection of His-tagged DYRK1A and Myc-tagged MDM2 or empty vector into U2OS cells, and cells were then treated with 50 μg/mL cycloheximide for the indicated durations before harvest. Protein levels of DYRK1A and MDM2 were analyzed by western blot. e Signals on the immunoblots of Fig. 5d were analyzed by the ImageJ software (NIH, Bethesda, MD, USA) 44 and the DYRK1A protein amounts were normalized with that of GAPDH. f Downregulation of DYRK1A by p53 activation requires MDM2. U2OS cells were transfected with small interfering RNA (siRNA) duplexes (200 nM) specific to MDM2 or negative oligo in serum-free medium for 4 h, and then were incubated with complete medium for 48 h. The protein levels of MDM2, p53, and DYRK1A in U2OS (siNeg and siMDM2) were measured 72 h after treatment with Nut3a. g Downregulation of DYRK1A by p53 activation requires MDM2, as determined by immunofluorescence. Scale bar, 10 μm. Right, quantitative immunointensity of DYRK1A was analyzed by the ImageJ software (NIH, Bethesda, MD, USA) *** p
    Figure Legend Snippet: Negative regulation of DYRK1A by p53 is mediated by MDM2. a Downregulation of dual-specificity tyrosine-phosphorylated and tyrosine-regulated kinase 1A (DYRK1A) by p53 activation is attenuated by MG132. U2OS and A2780 cells were treated with 8 μM Nut3a for 48 h, and were then treated with 10 μM MG132 for the last 6 h before harvest. Protein levels of p53, epidermal growth factor receptor (EGFR), and DYRK1A were analyzed by western blot. b Nut3a shortens the half-life of DYRK1A proteins. U2OS cells were treated with dimethyl sulfoxide (DMSO) or 8 μM Nut3a for 17 h, and were then treated with 50 μg/mL cycloheximide for the indicated durations before harvest. Protein levels of DYRK1A were analyzed by western blot. Bottom: Signals on the immunoblots were analyzed by the ImageJ software (NIH, Bethesda, MD, USA) 45 and the DYRK1A protein amounts were normalized with that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). c Ectopic expression of MDM2 downregulates DYRK1A. pCMV-myc3-HDM2 (WT) and MDM2 C464A expression vectors were respectively transfected into U2OS cells using Lipofectamine 2000, and cells transfected with empty vector were as control. After 48 h, the cells extracts were examined by western blot for the determination of MDM2, DYRK1A. d MDM2 shortens the half-life of DYRK1A proteins. Co-transfection of His-tagged DYRK1A and Myc-tagged MDM2 or empty vector into U2OS cells, and cells were then treated with 50 μg/mL cycloheximide for the indicated durations before harvest. Protein levels of DYRK1A and MDM2 were analyzed by western blot. e Signals on the immunoblots of Fig. 5d were analyzed by the ImageJ software (NIH, Bethesda, MD, USA) 44 and the DYRK1A protein amounts were normalized with that of GAPDH. f Downregulation of DYRK1A by p53 activation requires MDM2. U2OS cells were transfected with small interfering RNA (siRNA) duplexes (200 nM) specific to MDM2 or negative oligo in serum-free medium for 4 h, and then were incubated with complete medium for 48 h. The protein levels of MDM2, p53, and DYRK1A in U2OS (siNeg and siMDM2) were measured 72 h after treatment with Nut3a. g Downregulation of DYRK1A by p53 activation requires MDM2, as determined by immunofluorescence. Scale bar, 10 μm. Right, quantitative immunointensity of DYRK1A was analyzed by the ImageJ software (NIH, Bethesda, MD, USA) *** p

    Techniques Used: Activation Assay, Western Blot, Software, Expressing, Transfection, Plasmid Preparation, Cotransfection, Small Interfering RNA, Incubation, Immunofluorescence

    EGFR was downregulated by p53 activation in a subset of cancer cell lines. a , b Negative regulation of epidermal growth factor receptor (EGFR) by Nutlin-3a (Nut3a) in U87 cells. The protein levels of EGFR were determined by western blot ( a ) or by flow cytometry ( b , 8 μM for 72 h). Right, quantitative summary of EGFR expression by flow cytometry was shown. Representative results of three independent experiments were shown. c , d Negative regulation of EGFR by Nut3a in U2OS cells. c Western blot analysis of EGFR. d Flow cytometry analysis of EGFR in cells treated with Nut3a (8 μM) for 72 h. Right, quantitative summary of EGFR expression by flow cytometry. e Reduction of EGFR by Nut3a requires p53 function. Left, the protein levels of EGFR and p53 in U2OS cells transfected with pLKO.1-Puro-shNeg lentiviral vector or pLKO.1-Puro-shp53 lentiviral vector were measured 72 h after treatment with Nut3a. Right, U87 cells were transfected with small interfering RNA (siRNA) duplexes (200 nM) specific to p53 or negative oligo in serum-free medium for 4 h, and then were incubated with complete medium for 48 h. The protein levels of EGFR and p53 in U87 (siNeg and sip53) were measured 72 h after treatment with Nut3a (8 μM). f Downregulation of EGFR by ectopic expression of p53. p53 expression vectors were transfected into U2OS cells using Lipofectamine 2000, and cells transfected with empty vectors were as control. After 48 h, the cells extracts were examined by western blot for the determination of Flag, p53, EGFR. g , h EGFR was downregulated by Nut3a in HT1080, A172, and A2780 cells. The position of protein markers (in kDa) is indicated in each case. Immunoblots are representative of at least two independent experiments with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as a protein loading control. ** p
    Figure Legend Snippet: EGFR was downregulated by p53 activation in a subset of cancer cell lines. a , b Negative regulation of epidermal growth factor receptor (EGFR) by Nutlin-3a (Nut3a) in U87 cells. The protein levels of EGFR were determined by western blot ( a ) or by flow cytometry ( b , 8 μM for 72 h). Right, quantitative summary of EGFR expression by flow cytometry was shown. Representative results of three independent experiments were shown. c , d Negative regulation of EGFR by Nut3a in U2OS cells. c Western blot analysis of EGFR. d Flow cytometry analysis of EGFR in cells treated with Nut3a (8 μM) for 72 h. Right, quantitative summary of EGFR expression by flow cytometry. e Reduction of EGFR by Nut3a requires p53 function. Left, the protein levels of EGFR and p53 in U2OS cells transfected with pLKO.1-Puro-shNeg lentiviral vector or pLKO.1-Puro-shp53 lentiviral vector were measured 72 h after treatment with Nut3a. Right, U87 cells were transfected with small interfering RNA (siRNA) duplexes (200 nM) specific to p53 or negative oligo in serum-free medium for 4 h, and then were incubated with complete medium for 48 h. The protein levels of EGFR and p53 in U87 (siNeg and sip53) were measured 72 h after treatment with Nut3a (8 μM). f Downregulation of EGFR by ectopic expression of p53. p53 expression vectors were transfected into U2OS cells using Lipofectamine 2000, and cells transfected with empty vectors were as control. After 48 h, the cells extracts were examined by western blot for the determination of Flag, p53, EGFR. g , h EGFR was downregulated by Nut3a in HT1080, A172, and A2780 cells. The position of protein markers (in kDa) is indicated in each case. Immunoblots are representative of at least two independent experiments with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as a protein loading control. ** p

    Techniques Used: Activation Assay, Western Blot, Flow Cytometry, Cytometry, Expressing, Transfection, Plasmid Preparation, Small Interfering RNA, Incubation

    12) Product Images from "Effects of the Activin A–Follistatin System on Myocardial Cell Apoptosis through the Endoplasmic Reticulum Stress Pathway in Heart Failure"

    Article Title: Effects of the Activin A–Follistatin System on Myocardial Cell Apoptosis through the Endoplasmic Reticulum Stress Pathway in Heart Failure

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18020374

    Expression levels of activin A, follistatin, BNP, caspase-3 and ERS-related molecules in the left ventricular noninfarction area. ( A ) The expression of activin A, follistatin, BNP, caspase-3, glucose-regulated protein-78 (GRP-78), caspase-12, and CHOP was detected by Western blot assay; ( B ) The graph shows the result of densitometry quantification of proteins relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. ( a ) GRP-78, ( b ) Caspase-12, ( c ) CHOP, ( d ) Follistatin, ( e ) BNP, ( f ) activin A, ( g ) Caspase-3; ( C ) Activin A/Follistatin represents the ratio of activin A and follistatin. The data are presented as mean ± standard deviation. * p
    Figure Legend Snippet: Expression levels of activin A, follistatin, BNP, caspase-3 and ERS-related molecules in the left ventricular noninfarction area. ( A ) The expression of activin A, follistatin, BNP, caspase-3, glucose-regulated protein-78 (GRP-78), caspase-12, and CHOP was detected by Western blot assay; ( B ) The graph shows the result of densitometry quantification of proteins relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. ( a ) GRP-78, ( b ) Caspase-12, ( c ) CHOP, ( d ) Follistatin, ( e ) BNP, ( f ) activin A, ( g ) Caspase-3; ( C ) Activin A/Follistatin represents the ratio of activin A and follistatin. The data are presented as mean ± standard deviation. * p

    Techniques Used: Expressing, Western Blot, Standard Deviation

    Related Articles

    Diagnostic Assay:

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    Centrifugation:

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    Article Snippet: Cells extracts were performed in radioimmunoprecipitation assay (RIPA) buffer [100 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris pH 8] supplemented with protease (Roche) and phosphatase inhibitor cocktails (Sigma) and clarified by centrifugation. .. Proteins were analyzed by Western blot using the monoclonal anti-TWIST1 2C1a (Abcam), anti-p21CIP1 clone SX118 (Dako), and anti-HEB A9 (Santa Cruz Biotechnology) and the polyclonal anti-TWIST2 (proteintech), anti-E2A V18, anti-ITF2 K12, anti-H-RAS C20, anti-p15INK4B C20, anti-p16INK4A H156, p-histone H3 (Ser10)-R (Santa Cruz Biotechnology), and anti-GAPDH Abs16 (Millipore) antibodies and horseradish peroxidase–conjugated secondary antibodies (Dako).

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
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    Article Title: γ‐Secretase Dependent Nuclear Targeting of Dystroglycan
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    Autoradiography:

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    Immunostaining:

    Article Title: Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds, Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate, Chlorpromazine, and U18666A, in Prion-Infected Mouse Neuroblastoma Cells
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    Electrophoresis:

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
    Article Snippet: Equal amounts of extracted proteins were loaded and subjected to electrophoresis on 8% SDS-polyacrylamide gels (Beyotime, China) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, United States). .. Rabbit anti-GAPDH (1:10,000, Sigma, United States) was used as an internal loading control.

    Incubation:

    Article Title: Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization
    Article Snippet: Ten micrograms of total protein and 8 μ M fluorogenic γ -secretase substrate (Merck Millipore, Billerica, MA, USA) were incubated in assay buffer in a final reaction volume of 150 μ l for 5 h at 37 °C. .. Immunoblotting using rabbit-anti-GAPDH (Sigma-Aldrich; 1 h, RT) and densitometry were performed as described below.

    Article Title: ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1β activated endothelium in comparison to TNFα
    Article Snippet: Bands on membrane were visualised by chemiluminescence (Pierce ECL Plus Western Blotting Substrate, cat# 32132, Thermo Scientific) using ChemiDoc XRS+ Systems (Bio-Rad). .. Membranes were stripped, washed and blocked prior to incubation with rabbit anti-GAPDH (cat# 99545, Sigma, 1:5000) at 4°C overnight. .. Membranes were washed and incubated with Goat anti-Rabbit IgG-peroxidase (cat# A0545, Sigma, 1:16,000) for 2 h at room temperature, then visualised by chemiluminescence as described above.

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
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    Article Title: The LPS Responsiveness in BN and LEW Rats and Its Severity Are Modulated by the Liver
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    Article Title: p53 inhibits the expression of p125 and the methylation of POLD1 gene promoter by downregulating the Sp1-induced DNMT1 activities in breast cancer
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    Stripping Membranes:

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    Article Snippet: Immune complexes were detected by incubation with enhanced chemiluminescence Western blotting substrate (Thermo Fisher Scientific), followed by exposure to X-ray film. .. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibodies (Sigma, 1:2,000) were incubated with the same membrane following membrane stripping and re-staining. .. Sp1 antibody (Abcam, 10 μg) was diluted in 400 μL lysate/washing buffer (25 mM Tris [pH 7.4], 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% [v/v] Nonidet P-40, 5% [v/v] glycerol, and 0.25 mM phenylmethylsulfonyl fluoride).

    Activity Assay:

    Article Title: Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization
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    BIA-KA:

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
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    Acrylamide Gel Assay:

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    Western Blot:

    Article Title: Mitochondrial Peroxiredoxin 3 Regulates Sensory Cell Survival in the Cochlea
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    Article Title: The Heterodimeric TWIST1-E12 Complex Drives the Oncogenic Potential of TWIST1 in Human Mammary Epithelial Cells
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    Article Snippet: Paragraph title: Western blot ... Membranes were stripped, washed and blocked prior to incubation with rabbit anti-GAPDH (cat# 99545, Sigma, 1:5000) at 4°C overnight.

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
    Article Snippet: Paragraph title: Western Blot Analysis ... Rabbit anti-GAPDH (1:10,000, Sigma, United States) was used as an internal loading control.

    Article Title: The LPS Responsiveness in BN and LEW Rats and Its Severity Are Modulated by the Liver
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    Article Title: p53 inhibits the expression of p125 and the methylation of POLD1 gene promoter by downregulating the Sp1-induced DNMT1 activities in breast cancer
    Article Snippet: Paragraph title: Western blot analysis ... Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibodies (Sigma, 1:2,000) were incubated with the same membrane following membrane stripping and re-staining.

    Article Title: Ischemic preconditioning attenuates ischemia/reperfusion injury in rat steatotic liver: role of heme oxygenase-1-mediated autophagy
    Article Snippet: Paragraph title: Gel electrophoresis and western blotting ... The membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-HO-1(1:1000, Abcam, Cambridge, UK), rabbit anti-LC3B (1:1000, Abcam), rabbit anti-Atg16L1(1:1000, Abgent, San Diego, CA), rabbit anti-calpain2 (1:500, Cell Signaling Technology, Beverly, MA), rabbit anti-Atg7 (1:500, Cell Signaling Technology) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20000; Sigma-Aldrich).

    Immunohistochemistry:

    Article Title: Mitochondrial Peroxiredoxin 3 Regulates Sensory Cell Survival in the Cochlea
    Article Snippet: In addition, we address the potential regulation of Prx3 mRNA levels in explants of the mouse organ of Corti , . .. The primary antibodies used in this study are polyclonal rabbit anti-Prx3 for immunohistochemistry (ProteinTech Group Inc, Chicago, IL, # 10664-1-AP), monoclonal mouse anti-Prx3 for Western blotting (Abcam, Cambridge, UK, # ab16751), polyclonal rabbit anti-GAPDH (Millipore, Billerica, MA, # ABS16), and polyclonal rabbit anti-Myosin VII (Proteus Bioscience, Ramona, CA, # 25-6790). .. Prx3 siRNA (# MSS202040_a1N), goat anti-rabbit secondary antibody conjugated to Alexa Flour® 488, Alexa Fluor® 594, Hoechst 33342, 3′-(p -aminophenyl) fluorescein (APF), MitoTracker® Red CMXRos, BenchMark Prestained Protein Ladders, SuperScriptIII First-Strand Synthesis system for RT-PCR, Insulin-Transferrin-Selenium-A Supplement, and MasterMix were purchased from Invitrogen Life Technologies (Carlsbad, CA).

    Article Title: The Heterodimeric TWIST1-E12 Complex Drives the Oncogenic Potential of TWIST1 in Human Mammary Epithelial Cells
    Article Snippet: Paragraph title: Protein Analysis and Immunohistochemistry ... Proteins were analyzed by Western blot using the monoclonal anti-TWIST1 2C1a (Abcam), anti-p21CIP1 clone SX118 (Dako), and anti-HEB A9 (Santa Cruz Biotechnology) and the polyclonal anti-TWIST2 (proteintech), anti-E2A V18, anti-ITF2 K12, anti-H-RAS C20, anti-p15INK4B C20, anti-p16INK4A H156, p-histone H3 (Ser10)-R (Santa Cruz Biotechnology), and anti-GAPDH Abs16 (Millipore) antibodies and horseradish peroxidase–conjugated secondary antibodies (Dako).

    Protease Inhibitor:

    Article Title: Mitochondrial Peroxiredoxin 3 Regulates Sensory Cell Survival in the Cochlea
    Article Snippet: The primary antibodies used in this study are polyclonal rabbit anti-Prx3 for immunohistochemistry (ProteinTech Group Inc, Chicago, IL, # 10664-1-AP), monoclonal mouse anti-Prx3 for Western blotting (Abcam, Cambridge, UK, # ab16751), polyclonal rabbit anti-GAPDH (Millipore, Billerica, MA, # ABS16), and polyclonal rabbit anti-Myosin VII (Proteus Bioscience, Ramona, CA, # 25-6790). .. Secondary antibodies for Western blotting were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

    Article Title: Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome
    Article Snippet: Cells were lysed in lysis-buffer (Buffer: 10 mM HEPES, 1.5 mM MgCl2 , 300 mM sucrose, 10 mM KCl, 0.5% NP40, Roche Complete Protease Inhibitor Cocktail (Roche, Vienna, Austria), pH adjusted to 7.0, 5 mM DTT added freshly from a 1 M stock) for at least 1 h on ice. .. Protein was separated by SDS/PAGE, transferred to a nitrocellulose membrane and incubated with the relevant antibodies Caspase-2 (11B4) Alexis (Vienna, Austria); cleaved Caspase-3 (Asp175) (5A1), pCDK1 (Y15), Chk1 and p-Chk1 (S345; 133D3) all from Cell Signalling (New England Biolabs, Frankfurt, Germany); GAPDH (71.1) Sigma.

    Article Title: γ‐Secretase Dependent Nuclear Targeting of Dystroglycan
    Article Snippet: The pellet was resuspended in buffer 1 and mixed with an equal volume of buffer 2 (2 M sucrose, 10 mM Tris–HCl pH 8.0, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and complete protease inhibitor mixture). .. Use of antibodies to non‐phosphorylated β‐dystroglycan (MANDAG2) [Pereboev et al., ], β‐dystroglycan phosphorylated on tyrosine 892 (1709) [Thompson et al., ], and fractionation purity and loading control antibodies α‐tubulin (T5168), lamin A/C (4C11) and GAPDH (GA1R) (Sigma, Gillingham, UK) have also been described as above [Mathew et al., ].

    Cell Culture:

    Article Title: A Novel Lamin A Mutant Responsible for Congenital Muscular Dystrophy Causes Distinct Abnormalities of the Cell Nucleus
    Article Snippet: Subcutaneous fibroblasts obtained via skin biopsy from the patient (aged 16) and from two healthy individuals (her 43-year-old mother and an unrelated 18-year-old man) were cultured in low-glucose DMEM containing GlutaMAX™ and pyruvate (Gibco), and 10% foetal calf serum for experimental use between passages 6 and 14. .. Whole cell extracts of fibroblasts from controls and proband were analysed by western blot using antibodies directed against lamins (rabbit anti-lamin A/C and rabbit anti-lamin B1 as described in [ , ]) and GAPDH (rabbit anti-GAPDH, Sigma-Aldrich).

    Article Title: Ischemic preconditioning attenuates ischemia/reperfusion injury in rat steatotic liver: role of heme oxygenase-1-mediated autophagy
    Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-HO-1(1:1000, Abcam, Cambridge, UK), rabbit anti-LC3B (1:1000, Abcam), rabbit anti-Atg16L1(1:1000, Abgent, San Diego, CA), rabbit anti-calpain2 (1:500, Cell Signaling Technology, Beverly, MA), rabbit anti-Atg7 (1:500, Cell Signaling Technology) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20000; Sigma-Aldrich). .. The membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-HO-1(1:1000, Abcam, Cambridge, UK), rabbit anti-LC3B (1:1000, Abcam), rabbit anti-Atg16L1(1:1000, Abgent, San Diego, CA), rabbit anti-calpain2 (1:500, Cell Signaling Technology, Beverly, MA), rabbit anti-Atg7 (1:500, Cell Signaling Technology) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20000; Sigma-Aldrich).

    other:

    Article Title: Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac
    Article Snippet: Anti-FLAG rabbit polyclonal, anti-GAPDH rabbit polyclonal, anti-rabbit-Cy3, anti-mouse Alexa 594, and anti-rabbit Alexa 488 were obtained from Sigma.

    Imaging:

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
    Article Snippet: Rabbit anti-GAPDH (1:10,000, Sigma, United States) was used as an internal loading control. .. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies, including goat anti-rabbit IgG-HRP (Invitrogen, United States) and goat anti-chicken IgG-HRP (Invitrogen, United States), for 2 h at 4°C.

    Protein Concentration:

    Article Title: Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells
    Article Snippet: Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce). .. The blots were probed with anti-HO-1 (1:1000; Enzo Life Sciences, ADI-SPA-896, rabbit polyclonal), anti-Nrf2 (1:500; abcam, ab137550, rabbit polyclonal), anti-GAPDH (1:5000; Millipore, ABS16, rabbit polyclonal), anti-c-Myc (Wako, 017–2187, mouse monoclonal) or anti-Histone H1 (1:1000; Abcam, ab61177, rabbit polyclonal) antibodies.

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
    Article Snippet: The supernatant was collected, and the protein concentration was determined using the bicinchoninic acid (BCA) method with the PierceTM BCA Protein Assay Kit (Thermo Fisher, United States). .. Rabbit anti-GAPDH (1:10,000, Sigma, United States) was used as an internal loading control.

    Affinity Purification:

    Article Title: Pathomimetic avatars reveal divergent roles of microenvironment in invasive transition of ductal carcinoma in situ
    Article Snippet: Human cytokine antibody arrays (AAH-CYT-G5) were obtained from RayBiotech (Norcross, GA, USA). .. Human affinity-purified IL-6 neutralizing antibody (nAb; AF-206-NA) was purchased from R & D Systems (Minneapolis, MN, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) was obtained from EMD Millipore (Billerica, MA, USA). .. Monoclonal anti-CD10 antibody was purchased from Abcam (Cambridge, MA, USA).

    Recombinant:

    Article Title: Human Cytomegalovirus Infection Dysregulates the Canonical Wnt/?-catenin Signaling Pathway
    Article Snippet: Primary antibodies: mouse anti-β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA; 1∶500 dilution), mouse anti-β-actin (Abcam, Cambridge, MA; 1∶5,000), mouse anti-CMV IE1/2 (Millipore, Billerica, MA; 1∶200), mouse anti-CMV p65 (Santa Cruz Biotechnology; 1∶500), mouse anti-γ-tubulin (Abcam; 1∶1000), rabbit anti Histone 4 (H4) (Millipore, Billerica, MA; 1∶1000), rabbit anti-GAPDH (Sigma; 1∶5000), rabbit anti-caveolin-1 (Abcam; 1∶500). .. Primary antibodies: mouse anti-β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA; 1∶500 dilution), mouse anti-β-actin (Abcam, Cambridge, MA; 1∶5,000), mouse anti-CMV IE1/2 (Millipore, Billerica, MA; 1∶200), mouse anti-CMV p65 (Santa Cruz Biotechnology; 1∶500), mouse anti-γ-tubulin (Abcam; 1∶1000), rabbit anti Histone 4 (H4) (Millipore, Billerica, MA; 1∶1000), rabbit anti-GAPDH (Sigma; 1∶5000), rabbit anti-caveolin-1 (Abcam; 1∶500).

    Article Title: Pathomimetic avatars reveal divergent roles of microenvironment in invasive transition of ductal carcinoma in situ
    Article Snippet: Recombinant plasminogen activator inhibitor 1 (rPAI-1) protein, mutated to increase its stability, was purchased from EMD Millipore (Billerica, MA, USA). .. Human affinity-purified IL-6 neutralizing antibody (nAb; AF-206-NA) was purchased from R & D Systems (Minneapolis, MN, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) was obtained from EMD Millipore (Billerica, MA, USA).

    Immunofluorescence:

    Article Title: Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds, Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate, Chlorpromazine, and U18666A, in Prion-Infected Mouse Neuroblastoma Cells
    Article Snippet: Anti-Lamp1 rat mAb (Beckman Coulter, 1D4B), anti-sorting nexin 1 (Snx1) rabbit polyclonal antibodies (Proteintech Group, 10304-1-AP), anti-LC3 rabbit polyclonal antibodies (Medical & Biological Laboratories Co., Ltd, PM036), and anti-early endosome antigen 1 (EEA1) rabbit mAb (Cell Signaling Technology, C45B10) were used for IFA. .. Anti-β-actin mAb (Sigma, AC-15), anti-cathepsin D rabbit polyclonal antibodies (BioVision, 3191R-100) and anti-GAPDH rabbit polyclonal antibodies (Millipore, ABS16) were used for immunoblotting or dot-blotting.

    Nucleic Acid Electrophoresis:

    Article Title: Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization
    Article Snippet: For immunoblotting, 15 μ g of mammary tissue lysate homogenized in γ -secretase activity assay buffer was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% acrylamide gel. .. Immunoblotting using rabbit-anti-GAPDH (Sigma-Aldrich; 1 h, RT) and densitometry were performed as described below.

    Article Title: Ischemic preconditioning attenuates ischemia/reperfusion injury in rat steatotic liver: role of heme oxygenase-1-mediated autophagy
    Article Snippet: Paragraph title: Gel electrophoresis and western blotting ... The membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-HO-1(1:1000, Abcam, Cambridge, UK), rabbit anti-LC3B (1:1000, Abcam), rabbit anti-Atg16L1(1:1000, Abgent, San Diego, CA), rabbit anti-calpain2 (1:500, Cell Signaling Technology, Beverly, MA), rabbit anti-Atg7 (1:500, Cell Signaling Technology) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20000; Sigma-Aldrich).

    Bicinchoninic Acid Protein Assay:

    Article Title: Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells
    Article Snippet: Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce). .. The blots were probed with anti-HO-1 (1:1000; Enzo Life Sciences, ADI-SPA-896, rabbit polyclonal), anti-Nrf2 (1:500; abcam, ab137550, rabbit polyclonal), anti-GAPDH (1:5000; Millipore, ABS16, rabbit polyclonal), anti-c-Myc (Wako, 017–2187, mouse monoclonal) or anti-Histone H1 (1:1000; Abcam, ab61177, rabbit polyclonal) antibodies.

    Article Title: p53 inhibits the expression of p125 and the methylation of POLD1 gene promoter by downregulating the Sp1-induced DNMT1 activities in breast cancer
    Article Snippet: Protein concentrations were determined using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). .. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibodies (Sigma, 1:2,000) were incubated with the same membrane following membrane stripping and re-staining.

    Fluorescence:

    Article Title: Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization
    Article Snippet: To account for differences in cellular content in the mammary tissue from lactation to involution day 6, fluorescence was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level in homogenized mammary tissue samples as determined by immunoblotting ( ). .. Immunoblotting using rabbit-anti-GAPDH (Sigma-Aldrich; 1 h, RT) and densitometry were performed as described below.

    Isolation:

    Article Title: The LPS Responsiveness in BN and LEW Rats and Its Severity Are Modulated by the Liver
    Article Snippet: The total protein was isolated from liver tissues using RIPA buffer (Sigma-Aldrich, St. Louis, US). .. The membrane was incubated at 4°C overnight with primary antibodies: rabbit anti-phospho-AKT (Ser473, 1 : 1000), rabbit anti-phospho-Stat3 (Tyr705, 1 : 2000), rabbit anti-phospho-NF-κ B p65 (Ser536, 1 : 1000), rabbit anti-AKT (1 : 1000), rabbit anti-Stat3 (1 : 2000), anti-NF-κ B p65 (1 : 1000), rabbit anti-caspase 3 (1 : 1000), and rabbit anti-GAPDH (1 : 20000, Sigma-Aldrich, St. Louis, USA).

    Antibody Labeling:

    Article Title: Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds, Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate, Chlorpromazine, and U18666A, in Prion-Infected Mouse Neuroblastoma Cells
    Article Snippet: Anti-β-actin mAb (Sigma, AC-15), anti-cathepsin D rabbit polyclonal antibodies (BioVision, 3191R-100) and anti-GAPDH rabbit polyclonal antibodies (Millipore, ABS16) were used for immunoblotting or dot-blotting. .. Alexa Fluor 488-conjugated goat F(ab′)2 fragment anti-mouse IgG, Alexa Fluor 488- and 555-conjugated goat F(ab′)2 fragment anti-rabbit IgG and Alexa Fluor 555 conjugated goat IgG anti-rat IgG (Life Technologies) were used as secondary antibodies for IFA.

    Protein Extraction:

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
    Article Snippet: Briefly, protein extraction from peri-injury cortex tissues was performed by gently homogenizing the samples in RIPA lysis buffer with phosphatase inhibitors (Beyotime, China) with further centrifugation at 13,000 × g at 4°C for 20 min. .. Rabbit anti-GAPDH (1:10,000, Sigma, United States) was used as an internal loading control.

    Blocking Assay:

    Article Title: ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1β activated endothelium in comparison to TNFα
    Article Snippet: The blot was rinsed in TBST and was incubated in blocking buffer (10% non-fat milk in TBST) for 30 min with rocking. .. Membranes were stripped, washed and blocked prior to incubation with rabbit anti-GAPDH (cat# 99545, Sigma, 1:5000) at 4°C overnight.

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
    Article Snippet: Blocking buffer with 5% defatted milk was used to block the membranes for 1 h at room temperature, and membranes were incubated afterward with the following antibodies overnight at 4°C: rabbit anti-LRRK2 (1:10,000, Abcam, United States), rabbit anti-p38 (1:1000, Abcam, United States), rabbit anti-p-p38 (phospho-T180+Y182, 1:400, Abcam, United States), rabbit anti-Drosha (1:1000, Abcam, United States), and chicken anti-albumin (1:1000, Abcam, United States). .. Rabbit anti-GAPDH (1:10,000, Sigma, United States) was used as an internal loading control.

    Polyacrylamide Gel Electrophoresis:

    Article Title: p53 inhibits the expression of p125 and the methylation of POLD1 gene promoter by downregulating the Sp1-induced DNMT1 activities in breast cancer
    Article Snippet: Samples of cell lysate supernatant (20 μg protein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes. .. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibodies (Sigma, 1:2,000) were incubated with the same membrane following membrane stripping and re-staining.

    SDS Page:

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity
    Article Snippet: Protein lysates of MLECs or HLECs were prepared in ice‐cold radioimmune precipitation (RIPA) buffer, and SDS‐PAGE and protein blot analysis was performed as described , , . .. Anti‐rabbit glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) polyclonal Ab (Sigma‐Aldrich) was used to demonstrate that equal amounts of protein were loaded onto each lane.

    Article Title: Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization
    Article Snippet: For immunoblotting, 15 μ g of mammary tissue lysate homogenized in γ -secretase activity assay buffer was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% acrylamide gel. .. Immunoblotting using rabbit-anti-GAPDH (Sigma-Aldrich; 1 h, RT) and densitometry were performed as described below.

    Article Title: Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome
    Article Snippet: Cells were lysed in lysis-buffer (Buffer: 10 mM HEPES, 1.5 mM MgCl2 , 300 mM sucrose, 10 mM KCl, 0.5% NP40, Roche Complete Protease Inhibitor Cocktail (Roche, Vienna, Austria), pH adjusted to 7.0, 5 mM DTT added freshly from a 1 M stock) for at least 1 h on ice. .. Protein was separated by SDS/PAGE, transferred to a nitrocellulose membrane and incubated with the relevant antibodies Caspase-2 (11B4) Alexis (Vienna, Austria); cleaved Caspase-3 (Asp175) (5A1), pCDK1 (Y15), Chk1 and p-Chk1 (S345; 133D3) all from Cell Signalling (New England Biolabs, Frankfurt, Germany); GAPDH (71.1) Sigma. .. Immune-reactivity was visualized using an enhanced chemiluminescence detection system (ECL) (GE Healthcare, Freiburg, Germany).

    Article Title: γ‐Secretase Dependent Nuclear Targeting of Dystroglycan
    Article Snippet: The nuclear pellet was resuspended directly in SDS–PAGE sample buffer and used as the nuclear fraction in immunoblotting experiments. .. Use of antibodies to non‐phosphorylated β‐dystroglycan (MANDAG2) [Pereboev et al., ], β‐dystroglycan phosphorylated on tyrosine 892 (1709) [Thompson et al., ], and fractionation purity and loading control antibodies α‐tubulin (T5168), lamin A/C (4C11) and GAPDH (GA1R) (Sigma, Gillingham, UK) have also been described as above [Mathew et al., ].

    Plasmid Preparation:

    Article Title: Mitochondrial Peroxiredoxin 3 Regulates Sensory Cell Survival in the Cochlea
    Article Snippet: The primary antibodies used in this study are polyclonal rabbit anti-Prx3 for immunohistochemistry (ProteinTech Group Inc, Chicago, IL, # 10664-1-AP), monoclonal mouse anti-Prx3 for Western blotting (Abcam, Cambridge, UK, # ab16751), polyclonal rabbit anti-GAPDH (Millipore, Billerica, MA, # ABS16), and polyclonal rabbit anti-Myosin VII (Proteus Bioscience, Ramona, CA, # 25-6790). .. RIPA buffer was purchased from Cell Signaling Technology, Inc. (Danvers, MA), Complete Mini EDTA-free protease inhibitor cocktail tablets from Roche Diagnostic GmbH (Mannheim, Germany), RNeasy Micro Kit from Qiagen (Valencia, CA), and Enhanced Chemiluminescence Plus from Amersham Pharmacia Biotech (Piscataway, NJ).

    Software:

    Article Title: Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells
    Article Snippet: The blots were probed with anti-HO-1 (1:1000; Enzo Life Sciences, ADI-SPA-896, rabbit polyclonal), anti-Nrf2 (1:500; abcam, ab137550, rabbit polyclonal), anti-GAPDH (1:5000; Millipore, ABS16, rabbit polyclonal), anti-c-Myc (Wako, 017–2187, mouse monoclonal) or anti-Histone H1 (1:1000; Abcam, ab61177, rabbit polyclonal) antibodies. .. Immunoblot analysis was performed with horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG using enhanced chemiluminescence Western blotting detection reagents (Wako).

    Article Title: ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1β activated endothelium in comparison to TNFα
    Article Snippet: Membranes were stripped, washed and blocked prior to incubation with rabbit anti-GAPDH (cat# 99545, Sigma, 1:5000) at 4°C overnight. .. Membranes were washed and incubated with Goat anti-Rabbit IgG-peroxidase (cat# A0545, Sigma, 1:16,000) for 2 h at room temperature, then visualised by chemiluminescence as described above.

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
    Article Snippet: Rabbit anti-GAPDH (1:10,000, Sigma, United States) was used as an internal loading control. .. Immunoblots were finally probed with the ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore, United States) and visualized with an imaging system (Bio-Rad, United States).

    Article Title: Ischemic preconditioning attenuates ischemia/reperfusion injury in rat steatotic liver: role of heme oxygenase-1-mediated autophagy
    Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-HO-1(1:1000, Abcam, Cambridge, UK), rabbit anti-LC3B (1:1000, Abcam), rabbit anti-Atg16L1(1:1000, Abgent, San Diego, CA), rabbit anti-calpain2 (1:500, Cell Signaling Technology, Beverly, MA), rabbit anti-Atg7 (1:500, Cell Signaling Technology) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20000; Sigma-Aldrich). .. The membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-HO-1(1:1000, Abcam, Cambridge, UK), rabbit anti-LC3B (1:1000, Abcam), rabbit anti-Atg16L1(1:1000, Abgent, San Diego, CA), rabbit anti-calpain2 (1:500, Cell Signaling Technology, Beverly, MA), rabbit anti-Atg7 (1:500, Cell Signaling Technology) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20000; Sigma-Aldrich).

    Radio Immunoprecipitation:

    Article Title: The Heterodimeric TWIST1-E12 Complex Drives the Oncogenic Potential of TWIST1 in Human Mammary Epithelial Cells
    Article Snippet: Cells extracts were performed in radioimmunoprecipitation assay (RIPA) buffer [100 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris pH 8] supplemented with protease (Roche) and phosphatase inhibitor cocktails (Sigma) and clarified by centrifugation. .. Proteins were analyzed by Western blot using the monoclonal anti-TWIST1 2C1a (Abcam), anti-p21CIP1 clone SX118 (Dako), and anti-HEB A9 (Santa Cruz Biotechnology) and the polyclonal anti-TWIST2 (proteintech), anti-E2A V18, anti-ITF2 K12, anti-H-RAS C20, anti-p15INK4B C20, anti-p16INK4A H156, p-histone H3 (Ser10)-R (Santa Cruz Biotechnology), and anti-GAPDH Abs16 (Millipore) antibodies and horseradish peroxidase–conjugated secondary antibodies (Dako).

    Histone Deacetylase Assay:

    Article Title: A novel mechanism of ERK1/2 regulation in smooth muscle involving acetylation of the ERK1/2 scaffold IQGAP1
    Article Snippet: The Sirtuin inhibitor nicotinamide (NAM, Sigma) and the class I and II histone deacetylase (HDAC) inhibitor sodium phenylbutyrate (PB, Sigma) were both used at 5 mmol/L for 24 hours. .. For western blots, the following primary antibodies were used: rabbit polyclonal anti-pERK1/2 (1:2000, Cell Signaling, Danvers, MA), mouse monoclonal anti-ERK1/2 (1:500, Cell Signaling), mouse monoclonal anti-KSR1 (1:100, BD Biosciences, San Diego, CA), rabbit polyclonal anti-IQGAP1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-IQGAP1 (1:1000, Santa Cruz), goat polyclonal anti-IQGAP1 (1:500, Santa Cruz), rabbit polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:200,000, Sigma), rabbit polyclonal anti-acetylated lysine antibody (1:300, Cell Signaling), mouse monoclonal anti-tubulin (1:3000, Sigma).

    Concentration Assay:

    Article Title: γ‐Secretase Dependent Nuclear Targeting of Dystroglycan
    Article Snippet: Use of antibodies to non‐phosphorylated β‐dystroglycan (MANDAG2) [Pereboev et al., ], β‐dystroglycan phosphorylated on tyrosine 892 (1709) [Thompson et al., ], and fractionation purity and loading control antibodies α‐tubulin (T5168), lamin A/C (4C11) and GAPDH (GA1R) (Sigma, Gillingham, UK) have also been described as above [Mathew et al., ]. .. Optimal incubation times for Furin Inhibitor 1 and DAPT were determined in preliminary experiments to be 24 h (data not shown).

    Fractionation:

    Article Title: γ‐Secretase Dependent Nuclear Targeting of Dystroglycan
    Article Snippet: The retained supernatant was centrifuged at 9300g for 10 min at 4°C and the resultant supernatant was used as the cytoplasmic fraction [Mathew et al., ]. .. Use of antibodies to non‐phosphorylated β‐dystroglycan (MANDAG2) [Pereboev et al., ], β‐dystroglycan phosphorylated on tyrosine 892 (1709) [Thompson et al., ], and fractionation purity and loading control antibodies α‐tubulin (T5168), lamin A/C (4C11) and GAPDH (GA1R) (Sigma, Gillingham, UK) have also been described as above [Mathew et al., ]. .. Western blots were developed using enhanced chemiluminescence, imaged using a Biorad ChemiDoc WRX+ and quantified using Image Lab software (Hemel Hempstead, UK).

    Lysis:

    Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats
    Article Snippet: Briefly, protein extraction from peri-injury cortex tissues was performed by gently homogenizing the samples in RIPA lysis buffer with phosphatase inhibitors (Beyotime, China) with further centrifugation at 13,000 × g at 4°C for 20 min. .. Rabbit anti-GAPDH (1:10,000, Sigma, United States) was used as an internal loading control.

    Article Title: Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome
    Article Snippet: Cells were lysed in lysis-buffer (Buffer: 10 mM HEPES, 1.5 mM MgCl2 , 300 mM sucrose, 10 mM KCl, 0.5% NP40, Roche Complete Protease Inhibitor Cocktail (Roche, Vienna, Austria), pH adjusted to 7.0, 5 mM DTT added freshly from a 1 M stock) for at least 1 h on ice. .. Protein was separated by SDS/PAGE, transferred to a nitrocellulose membrane and incubated with the relevant antibodies Caspase-2 (11B4) Alexis (Vienna, Austria); cleaved Caspase-3 (Asp175) (5A1), pCDK1 (Y15), Chk1 and p-Chk1 (S345; 133D3) all from Cell Signalling (New England Biolabs, Frankfurt, Germany); GAPDH (71.1) Sigma.

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  • 99
    Millipore gapdh
    <t>S1PR3</t> is a target of miR-127 both in vitro and in vivo . ( a ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of BCL6 gene expression in TA muscle from 8-week-old WT and miR-127 TG mice. The data were normalized using <t>GAPDH</t> . BCL6 expression levels were further normalized to the expression level of WT, defined as 1. ( b ) Quantification of BCL6 mRNA in C2C12 cells grown in growth media (GM) and the indicated days during differentiation, determined by qRT-PCR. The data were normalized using GAPDH . BCL6 expression levels were further normalized to the expression level of GM, defined as 1. ( c ) Sequence alignment shows the target sites of miR-127 in the 3′-UTR of mouse S1PR3 , as predicted by miRWalk. A mutation in the seed matches is indicated by ‘mut'. ( d ) miR-127 directly repressed WT S1PR3 3′-UTR in luciferase assays in HEK293 cells, and the repression was abolished by mutation of the miR-127 binding site in the S1PR3 3′-UTR. The values are means±S.E. from three independent experiments. ( e ) Quantification of S1PR3 mRNA levels in miR-127-OE and control (NC) C2C12 cells by qRT-PCR. The data are a representative of three independent experiments, each performed in tripl icate. The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of NC, defined as 1. ( f ) S1PR3 mRNA levels in TA muscle from 8-week-old WT and miR-127 TG mice, determined by qRT-PCR ( n =5 mice per genotype). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of WT, defined as 1. ( g ) Western blot analysis of S1PR3 protein in TA muscles of 8-week-old WT and miR-127 TG mice. The numbers below each blot represent quantified signal intensities of individual bands. ( h ) qRT-PCR analysis of S1PR3 in TA muscle at the indicated days after CTX-induced injury ( n =3 mice per genotype). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of 0 days, defined as 1. ( i ) qRT-PCR analysis of miR-127-3p in the same samples described in panel h. The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of 0 days, defined as 1. Values in graphs represent means±S.E. * P
    Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse anti glyceraldehyde 3 phosphate dehydrogenase
    HGF does not mimic MSC-CM mediated cellular effects. ( A ) Determination of HGF protein content in stimulation media by means of ELISA revealing a robust increase upon MSC conditioning. ( B ) No difference regarding CNPase positivity was observed among OPCs grown in α -MEM in the absence or presence of recombinant HGF, whereas MSC-CM reproducibly increased CNPase expression. ( C–E ) Determination of transcript levels by means of quantitative real-time RT-PCR. No significant differences regarding Id2, Id4 and GFAP transcript levels were observed among OPCs grown in α-MEM in the absence or presence of recombinant HGF. MSC-CM treatment significantly reduced transcript levels of all three genes at all time points. ( F–K ) Anti-HGF antibody mediated depletion experiments revealed no effect on astrocyte (GFAP, Id2, Id4) and oligodendroglial/myelin (CGT, MBP, CNPase) gene expression levels. <t>Glyceraldehyde-3-phosphate</t> dehydrogenase (GAPDH) expression was used as reference gene. All data are shown as mean values ± SEM derived from n = 3 experiments for each analysis. t-test (ns: not significant, * P
    Mouse Anti Glyceraldehyde 3 Phosphate Dehydrogenase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti glyceraldehyde 3 phosphate dehydrogenase/product/Millipore
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    Millipore mouse anti glyceraldehyde 3 pdh gapdh monoclonal antibody unconjugated
    Mice transgenic for hc-Myc . (A) Example of a chimeric animal (chimerism of 60 to 70%) that was generated by injection of ES cell clone 14 into Balb/c blastocysts (day 3.5). (B) Chimeric animals were cross-bred to wildtype B6 mice yielding black progeny. Brother and sister matings of hc-Myc heterozygous mice (tested by PCR analysis) gave rise to hc-Myc homozygous offspring. The picture represents a PCR analysis of the genotypes of the offspring. For analysis genomic DNA was isolated from tail tissue. As controls genomic DNA of ES cell clones 1 and 14 was used. mu.c-Myc: PCR product of murine c-Myc . hc-Myc: PCR product of hc-Myc . (C) Detection of human c-MYC2 (hu. c-MYC, 62 kDa) in splenic cells of homozygous hc-Myc mice by Western blotting. In splenic cells of wildtype mice murine c-MYC2 was detected (mu. c-MYC, ca. 64 kDa). For positive control protein extracts from a human lymphoblastoid cell line (LCL 1.11) were used. <t>Glyceraldehyde-3-phosphat-dehydrogenase</t> <t>(GAPDH,</t> 36 kDa) was used as loading control.
    Mouse Anti Glyceraldehyde 3 Pdh Gapdh Monoclonal Antibody Unconjugated, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti glyceraldehyde 3 pdh gapdh monoclonal antibody unconjugated/product/Millipore
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    S1PR3 is a target of miR-127 both in vitro and in vivo . ( a ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of BCL6 gene expression in TA muscle from 8-week-old WT and miR-127 TG mice. The data were normalized using GAPDH . BCL6 expression levels were further normalized to the expression level of WT, defined as 1. ( b ) Quantification of BCL6 mRNA in C2C12 cells grown in growth media (GM) and the indicated days during differentiation, determined by qRT-PCR. The data were normalized using GAPDH . BCL6 expression levels were further normalized to the expression level of GM, defined as 1. ( c ) Sequence alignment shows the target sites of miR-127 in the 3′-UTR of mouse S1PR3 , as predicted by miRWalk. A mutation in the seed matches is indicated by ‘mut'. ( d ) miR-127 directly repressed WT S1PR3 3′-UTR in luciferase assays in HEK293 cells, and the repression was abolished by mutation of the miR-127 binding site in the S1PR3 3′-UTR. The values are means±S.E. from three independent experiments. ( e ) Quantification of S1PR3 mRNA levels in miR-127-OE and control (NC) C2C12 cells by qRT-PCR. The data are a representative of three independent experiments, each performed in tripl icate. The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of NC, defined as 1. ( f ) S1PR3 mRNA levels in TA muscle from 8-week-old WT and miR-127 TG mice, determined by qRT-PCR ( n =5 mice per genotype). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of WT, defined as 1. ( g ) Western blot analysis of S1PR3 protein in TA muscles of 8-week-old WT and miR-127 TG mice. The numbers below each blot represent quantified signal intensities of individual bands. ( h ) qRT-PCR analysis of S1PR3 in TA muscle at the indicated days after CTX-induced injury ( n =3 mice per genotype). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of 0 days, defined as 1. ( i ) qRT-PCR analysis of miR-127-3p in the same samples described in panel h. The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of 0 days, defined as 1. Values in graphs represent means±S.E. * P

    Journal: Cell Death & Disease

    Article Title: miR-127 enhances myogenic cell differentiation by targeting S1PR3

    doi: 10.1038/cddis.2017.128

    Figure Lengend Snippet: S1PR3 is a target of miR-127 both in vitro and in vivo . ( a ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of BCL6 gene expression in TA muscle from 8-week-old WT and miR-127 TG mice. The data were normalized using GAPDH . BCL6 expression levels were further normalized to the expression level of WT, defined as 1. ( b ) Quantification of BCL6 mRNA in C2C12 cells grown in growth media (GM) and the indicated days during differentiation, determined by qRT-PCR. The data were normalized using GAPDH . BCL6 expression levels were further normalized to the expression level of GM, defined as 1. ( c ) Sequence alignment shows the target sites of miR-127 in the 3′-UTR of mouse S1PR3 , as predicted by miRWalk. A mutation in the seed matches is indicated by ‘mut'. ( d ) miR-127 directly repressed WT S1PR3 3′-UTR in luciferase assays in HEK293 cells, and the repression was abolished by mutation of the miR-127 binding site in the S1PR3 3′-UTR. The values are means±S.E. from three independent experiments. ( e ) Quantification of S1PR3 mRNA levels in miR-127-OE and control (NC) C2C12 cells by qRT-PCR. The data are a representative of three independent experiments, each performed in tripl icate. The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of NC, defined as 1. ( f ) S1PR3 mRNA levels in TA muscle from 8-week-old WT and miR-127 TG mice, determined by qRT-PCR ( n =5 mice per genotype). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of WT, defined as 1. ( g ) Western blot analysis of S1PR3 protein in TA muscles of 8-week-old WT and miR-127 TG mice. The numbers below each blot represent quantified signal intensities of individual bands. ( h ) qRT-PCR analysis of S1PR3 in TA muscle at the indicated days after CTX-induced injury ( n =3 mice per genotype). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of 0 days, defined as 1. ( i ) qRT-PCR analysis of miR-127-3p in the same samples described in panel h. The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of 0 days, defined as 1. Values in graphs represent means±S.E. * P

    Article Snippet: Proteins in lysates were resolved by SDS-PAGE, and then immunoblotted using primary antibodies against MHC (MF20; Developmental Studies Hybridoma Bank (DSHB)), MyoG (F5D; DSHB), S1PR3 (Abcam, Cambridge, MA, USA) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Millipore, Bedford, MA, USA).

    Techniques: In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Mouse Assay, Sequencing, Mutagenesis, Luciferase, Binding Assay, Western Blot

    S1PR3 knockdown promotes C2C12 cell differentiation. ( a ) Efficiency of siRNA-mediated S1PR3 knockdown. Expression of S1PR3 in C2C12 cells was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of negative control (NC), defined as 1. ( b ) Immunostaining for MyoG (upper, green), MHC (lower, green) and DAPI (magenta) in C2C12 cells transfected with siRNAs against S1PR3 or a scrambled NC sequence. Cells were cultured in DM for 24 h for MyoG staining and 48 h for MHC staining. DAPI staining indicates nuclei. Scale bars, 50 μ m. ( c ) Expression of MyoG in C2C12 cells described in panel b was analyzed by qRT-PCR. The data were normalized using GAPDH . MyoG expression levels were further normalized to the expression level of NC, defined as 1. ( d ) The number of MyoG + cells in panel b was calculated. The data are representative of three independent experiments. For each experiment, 10 views were analyzed, and the data are presented as the number of MyoG + cells per view. ( e ) Expression of MHC in C2C12 cells described in panel b was analyzed by qRT-PCR. The data were normalized using GAPDH . MHC expression levels were further normalized to the expression level of NC, defined as 1. ( f ) The fusion index for differentiated C2C12 cells described in panel b was calculated. The data are representative of three independent experiments; 10 views were analyzed for each experiment. Statistical analysis in panel f was performed with nonparametric tests. * P

    Journal: Cell Death & Disease

    Article Title: miR-127 enhances myogenic cell differentiation by targeting S1PR3

    doi: 10.1038/cddis.2017.128

    Figure Lengend Snippet: S1PR3 knockdown promotes C2C12 cell differentiation. ( a ) Efficiency of siRNA-mediated S1PR3 knockdown. Expression of S1PR3 in C2C12 cells was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of negative control (NC), defined as 1. ( b ) Immunostaining for MyoG (upper, green), MHC (lower, green) and DAPI (magenta) in C2C12 cells transfected with siRNAs against S1PR3 or a scrambled NC sequence. Cells were cultured in DM for 24 h for MyoG staining and 48 h for MHC staining. DAPI staining indicates nuclei. Scale bars, 50 μ m. ( c ) Expression of MyoG in C2C12 cells described in panel b was analyzed by qRT-PCR. The data were normalized using GAPDH . MyoG expression levels were further normalized to the expression level of NC, defined as 1. ( d ) The number of MyoG + cells in panel b was calculated. The data are representative of three independent experiments. For each experiment, 10 views were analyzed, and the data are presented as the number of MyoG + cells per view. ( e ) Expression of MHC in C2C12 cells described in panel b was analyzed by qRT-PCR. The data were normalized using GAPDH . MHC expression levels were further normalized to the expression level of NC, defined as 1. ( f ) The fusion index for differentiated C2C12 cells described in panel b was calculated. The data are representative of three independent experiments; 10 views were analyzed for each experiment. Statistical analysis in panel f was performed with nonparametric tests. * P

    Article Snippet: Proteins in lysates were resolved by SDS-PAGE, and then immunoblotted using primary antibodies against MHC (MF20; Developmental Studies Hybridoma Bank (DSHB)), MyoG (F5D; DSHB), S1PR3 (Abcam, Cambridge, MA, USA) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Millipore, Bedford, MA, USA).

    Techniques: Cell Differentiation, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control, Immunostaining, Transfection, Sequencing, Cell Culture, Staining

    miR-127 ameliorates the dystrophic phenotype of mdx mice. ( a ) Fold overexpression of miR-127-3p in mdx;miR-127 mice, determined by quantitative real-time polymerase chain reaction (qRT-PCR). The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of WT, defined as 1. ( b ) S1PR3 mRNA levels in mdx;miR-127 mice, determined by qRT-PCR. The data were normalized using GAPDH. S1PR3 expression levels were further normalized to the expression level of WT, defined as 1. ( c ) Serum CK levels in WT, miR-127 TG, mdx , and mdx;miR-127 mice at 3 month of age. ( d ) EBD uptake in TA muscles of mdx and mdx;miR-127 mice at 3 month of age. EBD was detected as a red signal under a fluorescence microscope. Laminin immunostaining is shown in green. Scale bars, 100 μ m. ( e ) The percentages of EBD + fiber areas in TA muscles from mdx and mdx;miR- 127 mice. ( f ) Representative images of EBD + areas in quadriceps muscles of mdx and mdx;miR-127 mice at 3 month of age. EBD was detected as a red signal under a fluorescence microscope. Laminin immunostaining is shown in green. Scale bars, 100 μ m. ( g ) The percentages of EBD + fiber areas in quadriceps (QU) muscles from mdx and mdx;miR-127 mice. ( h ) Cross-sectional areas of myofibers with centralized nuclei in TA muscles from mdx and mdx;miR-127 mice ( n =5 mice per group). ( i ) Three-month-old WT, miR-127 TG, mdx , and mdx;miR-127 mice were subjected to forced downhill running on a treadmill. Muscle performance was measured as total running time to exhaustion. ( j and k ) EDL muscles isolated from WT, miR-127 TG, mdx , and mdx;miR-127 mice were electrically stimulated in vitro to elicit tetanic contractions. Maximal twitch force ( j ) and peak tetanic forces ( k ) were determined. Values are presented as means±S.E. ( n =5 mice per genotype). * P

    Journal: Cell Death & Disease

    Article Title: miR-127 enhances myogenic cell differentiation by targeting S1PR3

    doi: 10.1038/cddis.2017.128

    Figure Lengend Snippet: miR-127 ameliorates the dystrophic phenotype of mdx mice. ( a ) Fold overexpression of miR-127-3p in mdx;miR-127 mice, determined by quantitative real-time polymerase chain reaction (qRT-PCR). The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of WT, defined as 1. ( b ) S1PR3 mRNA levels in mdx;miR-127 mice, determined by qRT-PCR. The data were normalized using GAPDH. S1PR3 expression levels were further normalized to the expression level of WT, defined as 1. ( c ) Serum CK levels in WT, miR-127 TG, mdx , and mdx;miR-127 mice at 3 month of age. ( d ) EBD uptake in TA muscles of mdx and mdx;miR-127 mice at 3 month of age. EBD was detected as a red signal under a fluorescence microscope. Laminin immunostaining is shown in green. Scale bars, 100 μ m. ( e ) The percentages of EBD + fiber areas in TA muscles from mdx and mdx;miR- 127 mice. ( f ) Representative images of EBD + areas in quadriceps muscles of mdx and mdx;miR-127 mice at 3 month of age. EBD was detected as a red signal under a fluorescence microscope. Laminin immunostaining is shown in green. Scale bars, 100 μ m. ( g ) The percentages of EBD + fiber areas in quadriceps (QU) muscles from mdx and mdx;miR-127 mice. ( h ) Cross-sectional areas of myofibers with centralized nuclei in TA muscles from mdx and mdx;miR-127 mice ( n =5 mice per group). ( i ) Three-month-old WT, miR-127 TG, mdx , and mdx;miR-127 mice were subjected to forced downhill running on a treadmill. Muscle performance was measured as total running time to exhaustion. ( j and k ) EDL muscles isolated from WT, miR-127 TG, mdx , and mdx;miR-127 mice were electrically stimulated in vitro to elicit tetanic contractions. Maximal twitch force ( j ) and peak tetanic forces ( k ) were determined. Values are presented as means±S.E. ( n =5 mice per genotype). * P

    Article Snippet: Proteins in lysates were resolved by SDS-PAGE, and then immunoblotted using primary antibodies against MHC (MF20; Developmental Studies Hybridoma Bank (DSHB)), MyoG (F5D; DSHB), S1PR3 (Abcam, Cambridge, MA, USA) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Millipore, Bedford, MA, USA).

    Techniques: Mouse Assay, Over Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, Immunostaining, Isolation, In Vitro

    miR-127 enhances C2C12 cell differentiation. ( a ) Detection of the mature form of miR-127-3p by Northern blotting in the indicated tissues from 3-week-old mice. Transfer RNA (tRNA) was used as a loading control. ( b and c ) Quantification of miR-127-3p expression in C2C12 cells ( b ) and primary myoblasts ( c ) by quantitative real-time polymerase chain reaction (qRT-PCR) at the indicated days during differentiation. The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of 0 days, defined as 1. ( d ) Fold overexpression of miR-127-3p in C2C12 cells, quantified by qRT-PCR. OE, C2C12 cells infected with miR-127-expressing lentivirus; NC, C2C12 cells infected with control lentivirus. The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of NC, defined as 1. ( e ) Immunostaining for MyoG (green) and DAPI (magenta) after 24 h of culture in DM. Scale bars, 50 μ m. ( f ) Quantification of MyoG + cells presented in panel e. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed, and data are presented as positive cell numbers per view. Statistical analysis was performed with nonparametric tests. ( g ) Quantification of MyoG mRNA levels by qRT-PCR in NC and miR-127 OE cells, differentiated as in panel e. The data were normalized using GAPDH. MyoG expression levels were further normalized to the expression level of NC, defined as 1. Statistical analysis was performed with nonparametric tests. ( h ) Western blot analysis of MyoG protein levels in NC and miR-127 OE cells, differentiated as in panel e. GAPDH was used as a loading control. The numbers below each blot were the relative quantification of band intensity determined by Image J. ( i ) Immunostaining for MHC (green) and DAPI (magenta) after 48 h of culture in DM. Scale bars, 50 μ m. ( j ) Fusion index of the differentiated cells presented in panel I. The fusion index is calculated as the percentage of total nuclei present in multinucleated myotubes. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed. ( k ) Quantification of MHC mRNA levels by qRT-PCR in NC and miR-127 OE cells, differentiated as in panel I. The data were normalized using GAPDH. MHC expression levels were further normalized to the expression level of NC, defined as 1. ( l ) Western blot analysis of MHC protein levels in NC and miR-127 OE cells, differentiated as in panel ( i ). GAPDH was used as a loading control. The numbers below each blot were the relative quantification of band intensity determined by Image J. Value in graphs represent means±S.E. of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: miR-127 enhances myogenic cell differentiation by targeting S1PR3

    doi: 10.1038/cddis.2017.128

    Figure Lengend Snippet: miR-127 enhances C2C12 cell differentiation. ( a ) Detection of the mature form of miR-127-3p by Northern blotting in the indicated tissues from 3-week-old mice. Transfer RNA (tRNA) was used as a loading control. ( b and c ) Quantification of miR-127-3p expression in C2C12 cells ( b ) and primary myoblasts ( c ) by quantitative real-time polymerase chain reaction (qRT-PCR) at the indicated days during differentiation. The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of 0 days, defined as 1. ( d ) Fold overexpression of miR-127-3p in C2C12 cells, quantified by qRT-PCR. OE, C2C12 cells infected with miR-127-expressing lentivirus; NC, C2C12 cells infected with control lentivirus. The data were normalized using U6. miR-127 expression levels were further normalized to the expression level of NC, defined as 1. ( e ) Immunostaining for MyoG (green) and DAPI (magenta) after 24 h of culture in DM. Scale bars, 50 μ m. ( f ) Quantification of MyoG + cells presented in panel e. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed, and data are presented as positive cell numbers per view. Statistical analysis was performed with nonparametric tests. ( g ) Quantification of MyoG mRNA levels by qRT-PCR in NC and miR-127 OE cells, differentiated as in panel e. The data were normalized using GAPDH. MyoG expression levels were further normalized to the expression level of NC, defined as 1. Statistical analysis was performed with nonparametric tests. ( h ) Western blot analysis of MyoG protein levels in NC and miR-127 OE cells, differentiated as in panel e. GAPDH was used as a loading control. The numbers below each blot were the relative quantification of band intensity determined by Image J. ( i ) Immunostaining for MHC (green) and DAPI (magenta) after 48 h of culture in DM. Scale bars, 50 μ m. ( j ) Fusion index of the differentiated cells presented in panel I. The fusion index is calculated as the percentage of total nuclei present in multinucleated myotubes. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed. ( k ) Quantification of MHC mRNA levels by qRT-PCR in NC and miR-127 OE cells, differentiated as in panel I. The data were normalized using GAPDH. MHC expression levels were further normalized to the expression level of NC, defined as 1. ( l ) Western blot analysis of MHC protein levels in NC and miR-127 OE cells, differentiated as in panel ( i ). GAPDH was used as a loading control. The numbers below each blot were the relative quantification of band intensity determined by Image J. Value in graphs represent means±S.E. of three independent experiments. * P

    Article Snippet: Proteins in lysates were resolved by SDS-PAGE, and then immunoblotted using primary antibodies against MHC (MF20; Developmental Studies Hybridoma Bank (DSHB)), MyoG (F5D; DSHB), S1PR3 (Abcam, Cambridge, MA, USA) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Millipore, Bedford, MA, USA).

    Techniques: Cell Differentiation, Northern Blot, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Infection, Immunostaining, Western Blot

    miR-127 enhances myogenic differentiation by targeting S1PR3 . ( a ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of S1PR3 fold overexpression in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid; cells transfected with empty vector served as a negative control (NC). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. ( b ) Immunostaining for MyoG (green) in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid and NC cells transfected with empty vector. The cells were cultured in DM for 24 h. DAPI staining (magenta) indicates nuclei. Scale bars, 50 μ m. ( c ) Quantification of MyoG + cells in panel b. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed, and data are presented as positive cell numbers per view. ( d ) Quantification of MyoG expression by qRT-PCR in cells described in panel b. The data were normalized using GAPDH . MyoG expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. ( e ) Immunostaining for MHC (green) in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid and NC cells transfected with empty vector (NC). The cells were cultured in DM for 48 h. DAPI staining (magenta) indicates nuclei. Scale bars, 50 μ m. ( f ) The fusion index, calculated as the percentage of the total nuclei present in multinucleated myotubes, was determined for MHC-stained cells presented in panel e. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed. ( g ) Quantification of MHC expression in cells indicated in panel ( e ). The data were normalized using GAPDH . MHC expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. NC, C2C12 cells infected with control lentivirus; OE, C2C12 cells infected with miR-127-OE lentivirus. Values represent means±S.E. of at least three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: miR-127 enhances myogenic cell differentiation by targeting S1PR3

    doi: 10.1038/cddis.2017.128

    Figure Lengend Snippet: miR-127 enhances myogenic differentiation by targeting S1PR3 . ( a ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of S1PR3 fold overexpression in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid; cells transfected with empty vector served as a negative control (NC). The data were normalized using GAPDH . S1PR3 expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. ( b ) Immunostaining for MyoG (green) in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid and NC cells transfected with empty vector. The cells were cultured in DM for 24 h. DAPI staining (magenta) indicates nuclei. Scale bars, 50 μ m. ( c ) Quantification of MyoG + cells in panel b. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed, and data are presented as positive cell numbers per view. ( d ) Quantification of MyoG expression by qRT-PCR in cells described in panel b. The data were normalized using GAPDH . MyoG expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. ( e ) Immunostaining for MHC (green) in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid and NC cells transfected with empty vector (NC). The cells were cultured in DM for 48 h. DAPI staining (magenta) indicates nuclei. Scale bars, 50 μ m. ( f ) The fusion index, calculated as the percentage of the total nuclei present in multinucleated myotubes, was determined for MHC-stained cells presented in panel e. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed. ( g ) Quantification of MHC expression in cells indicated in panel ( e ). The data were normalized using GAPDH . MHC expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. NC, C2C12 cells infected with control lentivirus; OE, C2C12 cells infected with miR-127-OE lentivirus. Values represent means±S.E. of at least three independent experiments. * P

    Article Snippet: Proteins in lysates were resolved by SDS-PAGE, and then immunoblotted using primary antibodies against MHC (MF20; Developmental Studies Hybridoma Bank (DSHB)), MyoG (F5D; DSHB), S1PR3 (Abcam, Cambridge, MA, USA) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Millipore, Bedford, MA, USA).

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Transfection, Expressing, Plasmid Preparation, Negative Control, Immunostaining, Cell Culture, Staining, Infection

    Circadian oscillations and their LAN-induced circadian disruption in tumor signaling and transcriptional regulatory molecules involved in the Warburg effect (e.g., AKT, c-MYC and HIF-1α). ( A–F ) Tumor levels of phospho-AKT ser473 (A B), cMyc (C D), and HIF-1α (E F) were measured under LD,12∶12 or LD,12∶12+ LAN; see legend for Figure 1 for further experimental conditions. Solid black or red circles represent the mean ±SD relative expression (derived from the densitometric quantitation of the immunoblots) of either pAKT ser473 , c-MYC and HIF-1α at each circadian time point; n = 3 representative tumor samples at each time point. Relative expression of pAKT ser473 represents the ratio of pAKT ser473 protein to total (t)AKT protein; relative expression of c-MYC and HIF-1α represents the ratio of these proteins to tubulin and GAPDH, respectively. Cosinor analysis revealed robust and highly significant rhythmic patterns under LD,12∶12 conditions in tumor analytes; except for c-MYC, no significant daily rhythmic patterns were detected under dim LAN (see summary Table 4 ). Statistical comparisons were unable to be made between corresponding time points between the LD,12∶12 and LAN groups since immunoblots were performed for the two groups in separate runs. Only a single 24-hour cycle of immunoblots were run and displayed, whereas the densitometric representation of those same immunoblots was displayed twice to illustrate rhythm continuity as indicated in the legend of Fig. 1.

    Journal: PLoS ONE

    Article Title: Light Exposure at Night Disrupts Host/Cancer Circadian Regulatory Dynamics: Impact on the Warburg Effect, Lipid Signaling and Tumor Growth Prevention

    doi: 10.1371/journal.pone.0102776

    Figure Lengend Snippet: Circadian oscillations and their LAN-induced circadian disruption in tumor signaling and transcriptional regulatory molecules involved in the Warburg effect (e.g., AKT, c-MYC and HIF-1α). ( A–F ) Tumor levels of phospho-AKT ser473 (A B), cMyc (C D), and HIF-1α (E F) were measured under LD,12∶12 or LD,12∶12+ LAN; see legend for Figure 1 for further experimental conditions. Solid black or red circles represent the mean ±SD relative expression (derived from the densitometric quantitation of the immunoblots) of either pAKT ser473 , c-MYC and HIF-1α at each circadian time point; n = 3 representative tumor samples at each time point. Relative expression of pAKT ser473 represents the ratio of pAKT ser473 protein to total (t)AKT protein; relative expression of c-MYC and HIF-1α represents the ratio of these proteins to tubulin and GAPDH, respectively. Cosinor analysis revealed robust and highly significant rhythmic patterns under LD,12∶12 conditions in tumor analytes; except for c-MYC, no significant daily rhythmic patterns were detected under dim LAN (see summary Table 4 ). Statistical comparisons were unable to be made between corresponding time points between the LD,12∶12 and LAN groups since immunoblots were performed for the two groups in separate runs. Only a single 24-hour cycle of immunoblots were run and displayed, whereas the densitometric representation of those same immunoblots was displayed twice to illustrate rhythm continuity as indicated in the legend of Fig. 1.

    Article Snippet: The same blots were stripped and re-probed with antibodies to AKT (Cell Signaling Technology, Inc.), tubulin or GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Millipore Corp., Billerica, MA).

    Techniques: Expressing, Derivative Assay, Quantitation Assay, Western Blot

    Response of signaling and transcriptional regulatory factors involved in the Warburg effect (e.g., AKT, c-MYC and HIF-1α) in tissue-isolated SR- MCF-7 human breast cancer xenografts to short-term perfusion with nocturnal physiological concentrations of melatonin either in the absence or presence of melatonin receptor antagonist S20928 or 13-HODE. Xenografts were perfused in situ for 60 min with rat donor whole blood containing either vehicle, melatonin (500 pM or 1 nM), S20928 (1 nM), 13-HODE (12 µg/ml), melatonin+S20928 or melatonin +13-HODE. ( A – H ) Tumor perfusions were performed between 2–4 hours after lights on (0600 hours) under LD,12∶12 conditions. In the perfusions A B, a melatonin concentration of 1 nM was used whereas in all other perfusions (C – H) melatonin was used at 500 pM. Vertical bars represent the mean (±SD) relative expression (derived from the densitometric quantitation of the immunoblots) in tumors (n = 3–4) of either pAKT s473 (A–D), c-MYC (E F), or HIF-1α (G H). Relative expression of pAKT ser473 represents the ratio of pAKT ser473 protein to total (t)AKT protein; relative expression of c-MYC and HIF-1α represents the ratio of these proteins to GAPDH. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s Multiple Comparison Test to make multiple comparisons among all groups indicated by the brackets within each bar graph: * over each bracket indicates that differences are statistically significant at p

    Journal: PLoS ONE

    Article Title: Light Exposure at Night Disrupts Host/Cancer Circadian Regulatory Dynamics: Impact on the Warburg Effect, Lipid Signaling and Tumor Growth Prevention

    doi: 10.1371/journal.pone.0102776

    Figure Lengend Snippet: Response of signaling and transcriptional regulatory factors involved in the Warburg effect (e.g., AKT, c-MYC and HIF-1α) in tissue-isolated SR- MCF-7 human breast cancer xenografts to short-term perfusion with nocturnal physiological concentrations of melatonin either in the absence or presence of melatonin receptor antagonist S20928 or 13-HODE. Xenografts were perfused in situ for 60 min with rat donor whole blood containing either vehicle, melatonin (500 pM or 1 nM), S20928 (1 nM), 13-HODE (12 µg/ml), melatonin+S20928 or melatonin +13-HODE. ( A – H ) Tumor perfusions were performed between 2–4 hours after lights on (0600 hours) under LD,12∶12 conditions. In the perfusions A B, a melatonin concentration of 1 nM was used whereas in all other perfusions (C – H) melatonin was used at 500 pM. Vertical bars represent the mean (±SD) relative expression (derived from the densitometric quantitation of the immunoblots) in tumors (n = 3–4) of either pAKT s473 (A–D), c-MYC (E F), or HIF-1α (G H). Relative expression of pAKT ser473 represents the ratio of pAKT ser473 protein to total (t)AKT protein; relative expression of c-MYC and HIF-1α represents the ratio of these proteins to GAPDH. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s Multiple Comparison Test to make multiple comparisons among all groups indicated by the brackets within each bar graph: * over each bracket indicates that differences are statistically significant at p

    Article Snippet: The same blots were stripped and re-probed with antibodies to AKT (Cell Signaling Technology, Inc.), tubulin or GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Millipore Corp., Billerica, MA).

    Techniques: Isolation, In Situ, Concentration Assay, Expressing, Derivative Assay, Quantitation Assay, Western Blot

    HGF does not mimic MSC-CM mediated cellular effects. ( A ) Determination of HGF protein content in stimulation media by means of ELISA revealing a robust increase upon MSC conditioning. ( B ) No difference regarding CNPase positivity was observed among OPCs grown in α -MEM in the absence or presence of recombinant HGF, whereas MSC-CM reproducibly increased CNPase expression. ( C–E ) Determination of transcript levels by means of quantitative real-time RT-PCR. No significant differences regarding Id2, Id4 and GFAP transcript levels were observed among OPCs grown in α-MEM in the absence or presence of recombinant HGF. MSC-CM treatment significantly reduced transcript levels of all three genes at all time points. ( F–K ) Anti-HGF antibody mediated depletion experiments revealed no effect on astrocyte (GFAP, Id2, Id4) and oligodendroglial/myelin (CGT, MBP, CNPase) gene expression levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference gene. All data are shown as mean values ± SEM derived from n = 3 experiments for each analysis. t-test (ns: not significant, * P

    Journal: PLoS ONE

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation

    doi: 10.1371/journal.pone.0071814

    Figure Lengend Snippet: HGF does not mimic MSC-CM mediated cellular effects. ( A ) Determination of HGF protein content in stimulation media by means of ELISA revealing a robust increase upon MSC conditioning. ( B ) No difference regarding CNPase positivity was observed among OPCs grown in α -MEM in the absence or presence of recombinant HGF, whereas MSC-CM reproducibly increased CNPase expression. ( C–E ) Determination of transcript levels by means of quantitative real-time RT-PCR. No significant differences regarding Id2, Id4 and GFAP transcript levels were observed among OPCs grown in α-MEM in the absence or presence of recombinant HGF. MSC-CM treatment significantly reduced transcript levels of all three genes at all time points. ( F–K ) Anti-HGF antibody mediated depletion experiments revealed no effect on astrocyte (GFAP, Id2, Id4) and oligodendroglial/myelin (CGT, MBP, CNPase) gene expression levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference gene. All data are shown as mean values ± SEM derived from n = 3 experiments for each analysis. t-test (ns: not significant, * P

    Article Snippet: Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies.

    Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Quantitative RT-PCR, Derivative Assay

    Serum reduced conditioned medium. Determination of transcript levels by means of quantitative real-time RT-PCR. Investigation of GFAP, Id2 and Id4 gene expression levels in α -MEM versus MSC-CM containing 10% FBS ( A,C,E ). Investigation of GFAP, Id2 and Id4 gene expression levels in α -MEM versus MSC-CM containing 1% FBS ( B,D,F ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference gene; data are shown as mean values ± SEM derived from n = 7 for Id2, Id4 and n = 3 for GFAP ( A,C,E ) and n = 3 ( B,D,F ) experiments. t-test (ns: not significant, * P

    Journal: PLoS ONE

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation

    doi: 10.1371/journal.pone.0071814

    Figure Lengend Snippet: Serum reduced conditioned medium. Determination of transcript levels by means of quantitative real-time RT-PCR. Investigation of GFAP, Id2 and Id4 gene expression levels in α -MEM versus MSC-CM containing 10% FBS ( A,C,E ). Investigation of GFAP, Id2 and Id4 gene expression levels in α -MEM versus MSC-CM containing 1% FBS ( B,D,F ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference gene; data are shown as mean values ± SEM derived from n = 7 for Id2, Id4 and n = 3 for GFAP ( A,C,E ) and n = 3 ( B,D,F ) experiments. t-test (ns: not significant, * P

    Article Snippet: Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies.

    Techniques: Quantitative RT-PCR, Expressing, Derivative Assay

    MSC-CM leads to enhanced early myelin expression. Determination of transcript levels by means of quantitative real-time RT-PCR. ( A ) Upregulation of ceramide galactosyltransferase (CGT) expression was detected after three, six and nine days in culture, ( B ) whereas gene expression levels of CNPase were elevated at every measured time point. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference. ( C ) In addition, an increased percentage of CNPase-expressing OPCs was observed among MSC-CM treated cells as compared to cells grown in α -MEM. Significant differences were detected from day three onwards. ( D–E’’’ ) Representative immunofluorescent stainings of CNPase expressing OPCs at all four-time points of investigation. Data are shown as mean values ± SEM and derive from n = 8 (CGT), n = 8 (CNPase, q-RT-PCR) and n = 4 (CNPase, immunostainings) experiments. t-test (* P

    Journal: PLoS ONE

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation

    doi: 10.1371/journal.pone.0071814

    Figure Lengend Snippet: MSC-CM leads to enhanced early myelin expression. Determination of transcript levels by means of quantitative real-time RT-PCR. ( A ) Upregulation of ceramide galactosyltransferase (CGT) expression was detected after three, six and nine days in culture, ( B ) whereas gene expression levels of CNPase were elevated at every measured time point. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference. ( C ) In addition, an increased percentage of CNPase-expressing OPCs was observed among MSC-CM treated cells as compared to cells grown in α -MEM. Significant differences were detected from day three onwards. ( D–E’’’ ) Representative immunofluorescent stainings of CNPase expressing OPCs at all four-time points of investigation. Data are shown as mean values ± SEM and derive from n = 8 (CGT), n = 8 (CNPase, q-RT-PCR) and n = 4 (CNPase, immunostainings) experiments. t-test (* P

    Article Snippet: Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies.

    Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    Mice transgenic for hc-Myc . (A) Example of a chimeric animal (chimerism of 60 to 70%) that was generated by injection of ES cell clone 14 into Balb/c blastocysts (day 3.5). (B) Chimeric animals were cross-bred to wildtype B6 mice yielding black progeny. Brother and sister matings of hc-Myc heterozygous mice (tested by PCR analysis) gave rise to hc-Myc homozygous offspring. The picture represents a PCR analysis of the genotypes of the offspring. For analysis genomic DNA was isolated from tail tissue. As controls genomic DNA of ES cell clones 1 and 14 was used. mu.c-Myc: PCR product of murine c-Myc . hc-Myc: PCR product of hc-Myc . (C) Detection of human c-MYC2 (hu. c-MYC, 62 kDa) in splenic cells of homozygous hc-Myc mice by Western blotting. In splenic cells of wildtype mice murine c-MYC2 was detected (mu. c-MYC, ca. 64 kDa). For positive control protein extracts from a human lymphoblastoid cell line (LCL 1.11) were used. Glyceraldehyde-3-phosphat-dehydrogenase (GAPDH, 36 kDa) was used as loading control.

    Journal: PLoS ONE

    Article Title: Humanized c-Myc Mouse

    doi: 10.1371/journal.pone.0042021

    Figure Lengend Snippet: Mice transgenic for hc-Myc . (A) Example of a chimeric animal (chimerism of 60 to 70%) that was generated by injection of ES cell clone 14 into Balb/c blastocysts (day 3.5). (B) Chimeric animals were cross-bred to wildtype B6 mice yielding black progeny. Brother and sister matings of hc-Myc heterozygous mice (tested by PCR analysis) gave rise to hc-Myc homozygous offspring. The picture represents a PCR analysis of the genotypes of the offspring. For analysis genomic DNA was isolated from tail tissue. As controls genomic DNA of ES cell clones 1 and 14 was used. mu.c-Myc: PCR product of murine c-Myc . hc-Myc: PCR product of hc-Myc . (C) Detection of human c-MYC2 (hu. c-MYC, 62 kDa) in splenic cells of homozygous hc-Myc mice by Western blotting. In splenic cells of wildtype mice murine c-MYC2 was detected (mu. c-MYC, ca. 64 kDa). For positive control protein extracts from a human lymphoblastoid cell line (LCL 1.11) were used. Glyceraldehyde-3-phosphat-dehydrogenase (GAPDH, 36 kDa) was used as loading control.

    Article Snippet: Equal loading was confirmed by an antibody raised against glyceraldehyde-3-phosphat-dehydrogenase (MAB374, Millipore).

    Techniques: Mouse Assay, Transgenic Assay, Generated, Injection, Polymerase Chain Reaction, Isolation, Clone Assay, Western Blot, Positive Control

    Confirmation of homologous recombination and c-MYC2 expression in ES cell clones. (A) Genomic DNA of ES cell clones 1, 14, 18 and 19 and of wildtype ES cells (wt Bruce 4) was digested with Eco RI. Digested DNA was analyzed by Southern blotting with a 5′ probe and a 3′ probe. (wt) DNA fragment of the wildtype c-Myc locus; (rec.) DNA-fragment of recombined hc-Myc locus. (B) Protein extracts were prepared of ES cell clones 1, 14, 18 and 19 as well as of wildtype ES cells (wt Bruce 4) and of a human lymphoblastoid cell line (LCL 1.11). Human c-MYC2 (hu. c-MYC, ca. 62 kDa) was detected with antibody clone Y69. In wildtype ES cells murine c-MYC2 (mu. c-MYC, ca. 64 kDa) was detected. For loading control an antibody specific for glyceraldehyde-3-phosphat-dehydrogenase (GAPDH; ca. 36 kDa) was used. Western blot results were reproduced five times.

    Journal: PLoS ONE

    Article Title: Humanized c-Myc Mouse

    doi: 10.1371/journal.pone.0042021

    Figure Lengend Snippet: Confirmation of homologous recombination and c-MYC2 expression in ES cell clones. (A) Genomic DNA of ES cell clones 1, 14, 18 and 19 and of wildtype ES cells (wt Bruce 4) was digested with Eco RI. Digested DNA was analyzed by Southern blotting with a 5′ probe and a 3′ probe. (wt) DNA fragment of the wildtype c-Myc locus; (rec.) DNA-fragment of recombined hc-Myc locus. (B) Protein extracts were prepared of ES cell clones 1, 14, 18 and 19 as well as of wildtype ES cells (wt Bruce 4) and of a human lymphoblastoid cell line (LCL 1.11). Human c-MYC2 (hu. c-MYC, ca. 62 kDa) was detected with antibody clone Y69. In wildtype ES cells murine c-MYC2 (mu. c-MYC, ca. 64 kDa) was detected. For loading control an antibody specific for glyceraldehyde-3-phosphat-dehydrogenase (GAPDH; ca. 36 kDa) was used. Western blot results were reproduced five times.

    Article Snippet: Equal loading was confirmed by an antibody raised against glyceraldehyde-3-phosphat-dehydrogenase (MAB374, Millipore).

    Techniques: Homologous Recombination, Expressing, Clone Assay, Southern Blot, Western Blot