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Cell Signaling Technology Inc c ebp α
Effects of O. malacophyllus extracts on mRNA and protein expression of adipogenic transcription factors. 3T3-L1 cells were treated with O. malacophyllus extract (10, 50, and 100 μg/mL) during adipogenesis. The control was adipocytes differentiated for 8 days with MDI, a hormone mixture. NAC was the positive control. Total RNA was extracted on day 8, and ( A ) PPARγ, ( B ) <t>C/EBPα</t> and ( C ) aP2 mRNA were measured using quantitative real-time PCR. Total protein levels were measured using Western blotting with ( D ) PPARγ, ( E ) C/EBPα and ( F ) aP2 antibodies. mRNA and protein levels were quantified and normalized to that of β-actin. The band intensities were measured with software Imag. Each value represents the mean ± standard deviation of triplicate experiments. Values with different letters indicate statistically significant differences among groups at p
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1) Product Images from "Inhibitory effects of Orostachys malacophyllus var. iwarenge extracts on reactive oxygen species production and lipid accumulation during 3T3-L1 adipocyte differentiation"

Article Title: Inhibitory effects of Orostachys malacophyllus var. iwarenge extracts on reactive oxygen species production and lipid accumulation during 3T3-L1 adipocyte differentiation

Journal: Food Science and Biotechnology

doi: 10.1007/s10068-018-0426-x

Effects of O. malacophyllus extracts on mRNA and protein expression of adipogenic transcription factors. 3T3-L1 cells were treated with O. malacophyllus extract (10, 50, and 100 μg/mL) during adipogenesis. The control was adipocytes differentiated for 8 days with MDI, a hormone mixture. NAC was the positive control. Total RNA was extracted on day 8, and ( A ) PPARγ, ( B ) C/EBPα and ( C ) aP2 mRNA were measured using quantitative real-time PCR. Total protein levels were measured using Western blotting with ( D ) PPARγ, ( E ) C/EBPα and ( F ) aP2 antibodies. mRNA and protein levels were quantified and normalized to that of β-actin. The band intensities were measured with software Imag. Each value represents the mean ± standard deviation of triplicate experiments. Values with different letters indicate statistically significant differences among groups at p
Figure Legend Snippet: Effects of O. malacophyllus extracts on mRNA and protein expression of adipogenic transcription factors. 3T3-L1 cells were treated with O. malacophyllus extract (10, 50, and 100 μg/mL) during adipogenesis. The control was adipocytes differentiated for 8 days with MDI, a hormone mixture. NAC was the positive control. Total RNA was extracted on day 8, and ( A ) PPARγ, ( B ) C/EBPα and ( C ) aP2 mRNA were measured using quantitative real-time PCR. Total protein levels were measured using Western blotting with ( D ) PPARγ, ( E ) C/EBPα and ( F ) aP2 antibodies. mRNA and protein levels were quantified and normalized to that of β-actin. The band intensities were measured with software Imag. Each value represents the mean ± standard deviation of triplicate experiments. Values with different letters indicate statistically significant differences among groups at p

Techniques Used: Expressing, Positive Control, Real-time Polymerase Chain Reaction, Western Blot, Software, Standard Deviation

2) Product Images from "MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis"

Article Title: MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0045410

Adipogenic potency of SVSC and IVSC. The overall experimental scheme is illustrated in A. SVSC and IVSC differentiation was induced at d0, the cells (B-E, J represent SVSC; F-I, K represent IVSC; B-D and F-H, 100×; E and I 400×) were fixed and stained with oil red O at d3 (B; F,), d6 (C; G,) and d9 (D, E, J; H, I, K). Triglycerides contents in SVSC and IVSC in growth medium (GM) and differentiation medium (MDI) at d10 were also determined. Replicates were the same as L (L). Adipogenic-related mRNAs and miRNAs were quantified by qPCR at different times after MDI stimulation (M and N, respectively). Date were first normalized to GAPDH mRNA for each mRNA and miRNA, then fold changes relative to their respective levels in IVSC preadipocyte (day -2) were calculated, replicates were the same as L. C/EBPα protein levels during SVSC and IVSC differentiation were determined by western blotting, GAPDH protein was used as endogenous control (O).
Figure Legend Snippet: Adipogenic potency of SVSC and IVSC. The overall experimental scheme is illustrated in A. SVSC and IVSC differentiation was induced at d0, the cells (B-E, J represent SVSC; F-I, K represent IVSC; B-D and F-H, 100×; E and I 400×) were fixed and stained with oil red O at d3 (B; F,), d6 (C; G,) and d9 (D, E, J; H, I, K). Triglycerides contents in SVSC and IVSC in growth medium (GM) and differentiation medium (MDI) at d10 were also determined. Replicates were the same as L (L). Adipogenic-related mRNAs and miRNAs were quantified by qPCR at different times after MDI stimulation (M and N, respectively). Date were first normalized to GAPDH mRNA for each mRNA and miRNA, then fold changes relative to their respective levels in IVSC preadipocyte (day -2) were calculated, replicates were the same as L. C/EBPα protein levels during SVSC and IVSC differentiation were determined by western blotting, GAPDH protein was used as endogenous control (O).

Techniques Used: Staining, Real-time Polymerase Chain Reaction, Western Blot

QPCR validation of mRNAs and miRNAs post insulin stimulation during IVSC and SVSC adipogenesis. Eleven insulin-stimulated mRNAs and 8 miRNA listed in Fig. 9 were quantified during IVSC and SVSC differentiation. Data analysis and replicates were the same as Figure 1 . Wnt10b , C/EBPα , C/EBPβ , PPARγ1 , PPARγ2 are shown in Fig. 1M, and the others are shown in A. Some other mRNAs related to adipogenesis were selected and quantified by qPCR (B). MiR-27a, miR-27b and miR-210 are shown in Fig. 1N, and the others are shown in C. The adipogenic- related miR-143-3p was also quantified.
Figure Legend Snippet: QPCR validation of mRNAs and miRNAs post insulin stimulation during IVSC and SVSC adipogenesis. Eleven insulin-stimulated mRNAs and 8 miRNA listed in Fig. 9 were quantified during IVSC and SVSC differentiation. Data analysis and replicates were the same as Figure 1 . Wnt10b , C/EBPα , C/EBPβ , PPARγ1 , PPARγ2 are shown in Fig. 1M, and the others are shown in A. Some other mRNAs related to adipogenesis were selected and quantified by qPCR (B). MiR-27a, miR-27b and miR-210 are shown in Fig. 1N, and the others are shown in C. The adipogenic- related miR-143-3p was also quantified.

Techniques Used: Real-time Polymerase Chain Reaction

MiRNAs that regulate insulin-stimulated adipogenesis. A reverse search for miRNAs regulating mRNAs important in preadipocyte differentiation, lipogenesis and antilipolysis was performed. The relations between miRNAs and mRNAs were illustrated. INSR : insulin receptor; IRS1/2: insulin receptor substrate 1/2; PI3K : phosphoinositide-3-kinase, catalytic, polypeptide; PIP3 : interaction protein for cytohesin exchange factors 1; PDK1/2 : pyruvate dehydrogenase kinase, isozyme 1/2; S6k1 : ribosomal protein S6 kinase, polypeptide 1; aPKC : protein kinase C, alpha; SREBP1 : sterol regulatory element binding transcription factor 1; AKT : v-akt murine thymoma viral oncogene homolog; TSC2 : tuberous sclerosis 2; Rheb : Ras homolog enriched in brain; NEcdin : necdin homolog (mouse); CREB : cAMP responsive element binding protein, FOXO1 : RNA binding protein, fox-1 homolog 1; FOXOA2 : forkhead box A2; GATA2/3 : GATA binding protein 2/3; PPARγ : peroxisome proliferator-activated receptor gamma; C/EBPα/β/γ/δ/ζ : CCAAT/enhancer binding protein alpha/beta/gamma/delta/zeta; KROX20 : early growth response 2, KLF2/5 : Kruppel-like factor 2/5 (lung); wnt10b : wingless-type MMTV integration site family, member 10b; β-catenin : (cadherin-associated protein), beta 1; Glut4 : solute carrier family 2 (facilitated glucose transporter), member 4; GK : glycerol kinase; ACLY : ATP citrate lyase; ACAC : acetyl-CoA carboxylase; FASN : fatty acid synthase; SCD : stearoyl-CoA desaturase (delta-9-desaturase); GPAT2 : glycerol-3-phosphate acyltransferase 2, mitochondrial; PDE3 : phosphodiesterase 3, cGMP-inhibited; PRKAC : protein kinase, cAMP-dependent, catalytic; HSL : lipase, hormone-sensitive.
Figure Legend Snippet: MiRNAs that regulate insulin-stimulated adipogenesis. A reverse search for miRNAs regulating mRNAs important in preadipocyte differentiation, lipogenesis and antilipolysis was performed. The relations between miRNAs and mRNAs were illustrated. INSR : insulin receptor; IRS1/2: insulin receptor substrate 1/2; PI3K : phosphoinositide-3-kinase, catalytic, polypeptide; PIP3 : interaction protein for cytohesin exchange factors 1; PDK1/2 : pyruvate dehydrogenase kinase, isozyme 1/2; S6k1 : ribosomal protein S6 kinase, polypeptide 1; aPKC : protein kinase C, alpha; SREBP1 : sterol regulatory element binding transcription factor 1; AKT : v-akt murine thymoma viral oncogene homolog; TSC2 : tuberous sclerosis 2; Rheb : Ras homolog enriched in brain; NEcdin : necdin homolog (mouse); CREB : cAMP responsive element binding protein, FOXO1 : RNA binding protein, fox-1 homolog 1; FOXOA2 : forkhead box A2; GATA2/3 : GATA binding protein 2/3; PPARγ : peroxisome proliferator-activated receptor gamma; C/EBPα/β/γ/δ/ζ : CCAAT/enhancer binding protein alpha/beta/gamma/delta/zeta; KROX20 : early growth response 2, KLF2/5 : Kruppel-like factor 2/5 (lung); wnt10b : wingless-type MMTV integration site family, member 10b; β-catenin : (cadherin-associated protein), beta 1; Glut4 : solute carrier family 2 (facilitated glucose transporter), member 4; GK : glycerol kinase; ACLY : ATP citrate lyase; ACAC : acetyl-CoA carboxylase; FASN : fatty acid synthase; SCD : stearoyl-CoA desaturase (delta-9-desaturase); GPAT2 : glycerol-3-phosphate acyltransferase 2, mitochondrial; PDE3 : phosphodiesterase 3, cGMP-inhibited; PRKAC : protein kinase, cAMP-dependent, catalytic; HSL : lipase, hormone-sensitive.

Techniques Used: Binding Assay, RNA Binding Assay

3) Product Images from "Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition"

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition

Journal: Development (Cambridge, England)

doi: 10.1242/dev.168609

Compromised mitochondrial function contributes to adipose-like phenotype in injured neonatal mouse heart. (A) qPCR of Pparg in control and Pitx2 null P19 cell lines with or without 8 h of H 2 O 2 treatment. (B) Seahorse assay shows oxygen consumption rate (OCR) of control and P19- Pitx2 null cell lines with readout normalized to the cell number in each assay. The x -axis indicates measurements 1-11. The dashed vertical lines indicate injections into media of the specific stressors oligomycin, FCCP and antimycin A/rotenone (A/R). (C) Western blot shows expression of ETC components in control and Pit x 2 null P19 cell lines. (D,E) Trichrome staining of Cox7c lacZ/+ and control mouse hearts that were subjected to LAD occlusion at P2 and analyzed at 60 days after occlusion. (F) Quantification of the amount of scarring seen in D,E. (G) Echocardiography shows the ejection fraction of control and Cox7c lacZ/+ mice at 60 days after sham or LAD occlusion surgery. (H-J) Immunostaining for cTnT (green), C/EBPα (red) and DAPI (blue) in control (H) and Cox7c lacZ/+ (I) hearts at 60 days after LAD occlusion. Asterisks indicate scar area. Arrows indicate C/EBPα positive cells. The percentage of C/EBPα + scar cells is quantified in J. * P
Figure Legend Snippet: Compromised mitochondrial function contributes to adipose-like phenotype in injured neonatal mouse heart. (A) qPCR of Pparg in control and Pitx2 null P19 cell lines with or without 8 h of H 2 O 2 treatment. (B) Seahorse assay shows oxygen consumption rate (OCR) of control and P19- Pitx2 null cell lines with readout normalized to the cell number in each assay. The x -axis indicates measurements 1-11. The dashed vertical lines indicate injections into media of the specific stressors oligomycin, FCCP and antimycin A/rotenone (A/R). (C) Western blot shows expression of ETC components in control and Pit x 2 null P19 cell lines. (D,E) Trichrome staining of Cox7c lacZ/+ and control mouse hearts that were subjected to LAD occlusion at P2 and analyzed at 60 days after occlusion. (F) Quantification of the amount of scarring seen in D,E. (G) Echocardiography shows the ejection fraction of control and Cox7c lacZ/+ mice at 60 days after sham or LAD occlusion surgery. (H-J) Immunostaining for cTnT (green), C/EBPα (red) and DAPI (blue) in control (H) and Cox7c lacZ/+ (I) hearts at 60 days after LAD occlusion. Asterisks indicate scar area. Arrows indicate C/EBPα positive cells. The percentage of C/EBPα + scar cells is quantified in J. * P

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

4) Product Images from "Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2"

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2

Journal: Molecules

doi: 10.3390/molecules200815359

PPAR-γ2, C/EBP-α, adiponectin, SREBP-1 and FAS mRNA expression pattern in differentiated adipocytes were analyzed by qPCR. Data were shown as means ± SEM of six replicates. Different letters a, b, c, d within a treatment indicate a significant difference ( p
Figure Legend Snippet: PPAR-γ2, C/EBP-α, adiponectin, SREBP-1 and FAS mRNA expression pattern in differentiated adipocytes were analyzed by qPCR. Data were shown as means ± SEM of six replicates. Different letters a, b, c, d within a treatment indicate a significant difference ( p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Immunoblot analyses of PPAR-γ2, C/EBP-α, adiponectin, SREBP-1, and FAS. Bar diagram indicates fold expression of proteins (arbitrary units-AU) in differentiated adipocytes quantified by the Imagej software. Data were shown as means ± SEM of three replicates. Different letters a, b, c, d within a treatment indicate a significant difference ( p
Figure Legend Snippet: Immunoblot analyses of PPAR-γ2, C/EBP-α, adiponectin, SREBP-1, and FAS. Bar diagram indicates fold expression of proteins (arbitrary units-AU) in differentiated adipocytes quantified by the Imagej software. Data were shown as means ± SEM of three replicates. Different letters a, b, c, d within a treatment indicate a significant difference ( p

Techniques Used: Expressing, Software

5) Product Images from "Antiobesity Effect of Astilbe chinensis Franch. et Savet. Extract through Regulation of Adipogenesis and AMP-Activated Protein Kinase Pathways in 3T3-L1 Adipocyte and High-Fat Diet-Induced C57BL/6N Obese Mice"

Article Title: Antiobesity Effect of Astilbe chinensis Franch. et Savet. Extract through Regulation of Adipogenesis and AMP-Activated Protein Kinase Pathways in 3T3-L1 Adipocyte and High-Fat Diet-Induced C57BL/6N Obese Mice

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/1347612

Effects of Astilbe chinensis Franch. et Savat. (AC) extract on epididymal fat size in HFD induced obese mice. (a) The H E stained epididymal fat tissue from HFD induced obese mice treated with RD or AC (100 and 200 mg/kg), and (b) quantification of epididymal fat tissues size were calculated. The (c) mRNA levels of PPAR- γ , C/EBP α , SREBP1, FAS, and SCD-1, as determined by reverse transcription PCR (upper panels) and real-time PCR (lower panels), and (d) the protein levels of PPAR- γ , C/EBP α , SREBP1, FAS, and SCD-1 were examined by western blot analyses, respectively. ### p
Figure Legend Snippet: Effects of Astilbe chinensis Franch. et Savat. (AC) extract on epididymal fat size in HFD induced obese mice. (a) The H E stained epididymal fat tissue from HFD induced obese mice treated with RD or AC (100 and 200 mg/kg), and (b) quantification of epididymal fat tissues size were calculated. The (c) mRNA levels of PPAR- γ , C/EBP α , SREBP1, FAS, and SCD-1, as determined by reverse transcription PCR (upper panels) and real-time PCR (lower panels), and (d) the protein levels of PPAR- γ , C/EBP α , SREBP1, FAS, and SCD-1 were examined by western blot analyses, respectively. ### p

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

Effects of the Astilbe chinensis Franch. et Savat. (AC) extract on inhibiting 3T3-L1 adipocyte differentiation. (a) The 3T3-L1 cell proliferation was measured by MTS assay after 24 h, 48 h, 72 h, and 96 h treatment of AC extract at various concentrations. (b) The 3T3-L1 cells differentiated with differentiation medium in the absence or presence of AC extract for 8 days. Intracellular lipids were stained with Oil Red O, and triglyceride content was quantified by spectrometry at 540 nm. (c) mRNA levels of PPAR- γ , C/EBP α , SREBP1, FAS, and SCD-1, as determined by reverse transcription PCR (upper panels) and real-time PCR (lower panels), and (d) the protein expression levels of PPAR- γ , C/EBP α , SREBP1, FAS, and SCD-1 were examined by western blot analyses. Data are presented as the mean ± SEM of three independent experiments, each performed in triplicate. ### p
Figure Legend Snippet: Effects of the Astilbe chinensis Franch. et Savat. (AC) extract on inhibiting 3T3-L1 adipocyte differentiation. (a) The 3T3-L1 cell proliferation was measured by MTS assay after 24 h, 48 h, 72 h, and 96 h treatment of AC extract at various concentrations. (b) The 3T3-L1 cells differentiated with differentiation medium in the absence or presence of AC extract for 8 days. Intracellular lipids were stained with Oil Red O, and triglyceride content was quantified by spectrometry at 540 nm. (c) mRNA levels of PPAR- γ , C/EBP α , SREBP1, FAS, and SCD-1, as determined by reverse transcription PCR (upper panels) and real-time PCR (lower panels), and (d) the protein expression levels of PPAR- γ , C/EBP α , SREBP1, FAS, and SCD-1 were examined by western blot analyses. Data are presented as the mean ± SEM of three independent experiments, each performed in triplicate. ### p

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

6) Product Images from "miR-148a is Associated with Obesity and Modulates Adipocyte Differentiation of Mesenchymal Stem Cells through Wnt Signaling"

Article Title: miR-148a is Associated with Obesity and Modulates Adipocyte Differentiation of Mesenchymal Stem Cells through Wnt Signaling

Journal: Scientific Reports

doi: 10.1038/srep09930

Overexpression of miR-148a in hMSCs-Ad enhances adipogenic differentiation. (A) miR-148a overexpression efficiency was verified by qRT-PCR at Day 0. (B) Oil red O staining indicated the effect of miR-148a overexpression on hMSCs-Ad adipogenic differentiation at Days 1, 7, and 10. (C) Triacylglycerol content and GPDH activity (D) detected neutral lipid accumulation. (E) miR-148a overexpression in hMSCs-Ad promoted adipogenic maker gene transcription based on qRT-PCR at Days 0, 4, 7, and 10 during adipogenesis. (F) PPARγ2 and C/EBP-α protein levels were detected by Western blot under the same experimental conditions. Related mRNA levels are represented as mean ± SEM from three or more independent repeats. * P
Figure Legend Snippet: Overexpression of miR-148a in hMSCs-Ad enhances adipogenic differentiation. (A) miR-148a overexpression efficiency was verified by qRT-PCR at Day 0. (B) Oil red O staining indicated the effect of miR-148a overexpression on hMSCs-Ad adipogenic differentiation at Days 1, 7, and 10. (C) Triacylglycerol content and GPDH activity (D) detected neutral lipid accumulation. (E) miR-148a overexpression in hMSCs-Ad promoted adipogenic maker gene transcription based on qRT-PCR at Days 0, 4, 7, and 10 during adipogenesis. (F) PPARγ2 and C/EBP-α protein levels were detected by Western blot under the same experimental conditions. Related mRNA levels are represented as mean ± SEM from three or more independent repeats. * P

Techniques Used: Over Expression, Quantitative RT-PCR, Staining, Activity Assay, Western Blot

miR-148a regulates adipogenesis in hMSCs-Ad via Wnt. (A) Wnt signal pathway protein level was detected by Western blot. (B) Western blot analysis of Wnt1 in hMSCs-Ad lysates after stable infection with Wnt1-shRNA or Wnt1 construct or control lentivirus. GAPDH blot served as loading control. (C) Oil red O staining indicated the effects of miR-148a on hMSCs-Ad adipogenic differentiation at Days 4, 7, and 10. Transcription levels of adipogenic marker genes, PPARγ2 (D, G), C/EBP-α (E, H), and FABP4 (F, I), were detected by qRT-PCR in different groups at Days 0, 4, 7, and 10 during adipogenesis. * P
Figure Legend Snippet: miR-148a regulates adipogenesis in hMSCs-Ad via Wnt. (A) Wnt signal pathway protein level was detected by Western blot. (B) Western blot analysis of Wnt1 in hMSCs-Ad lysates after stable infection with Wnt1-shRNA or Wnt1 construct or control lentivirus. GAPDH blot served as loading control. (C) Oil red O staining indicated the effects of miR-148a on hMSCs-Ad adipogenic differentiation at Days 4, 7, and 10. Transcription levels of adipogenic marker genes, PPARγ2 (D, G), C/EBP-α (E, H), and FABP4 (F, I), were detected by qRT-PCR in different groups at Days 0, 4, 7, and 10 during adipogenesis. * P

Techniques Used: Western Blot, Infection, shRNA, Construct, Staining, Marker, Quantitative RT-PCR

7) Product Images from "Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach"

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach

Journal: Biological Research

doi: 10.1186/s40659-016-0098-z

Effects of EECV on PPAR-γ2, C/EBP-α and AMPK-α protein expression. EECV treatment upregulates PPAR-γ2, C/EBP-α and downregulates AMPK-α protein level on the 10th day. The results represent the mean ± SEM of three replicates different letters a , b , c , within a treatment indicates significant differences (p
Figure Legend Snippet: Effects of EECV on PPAR-γ2, C/EBP-α and AMPK-α protein expression. EECV treatment upregulates PPAR-γ2, C/EBP-α and downregulates AMPK-α protein level on the 10th day. The results represent the mean ± SEM of three replicates different letters a , b , c , within a treatment indicates significant differences (p

Techniques Used: Expressing

Impact of EECV on adipogenic and lipogenic mRNA expression quantified by qPCR. EECV treatment increased PPAR-γ2, C/EBP-α, adiponectin, FAS and leptin mRNA expression in a dose dependent manner on the 5th and 10th day of differentiation. The results represent the mean ± SEM of six replicates. Different letters a , b , c , d , within a treatment indicates significant differences (p
Figure Legend Snippet: Impact of EECV on adipogenic and lipogenic mRNA expression quantified by qPCR. EECV treatment increased PPAR-γ2, C/EBP-α, adiponectin, FAS and leptin mRNA expression in a dose dependent manner on the 5th and 10th day of differentiation. The results represent the mean ± SEM of six replicates. Different letters a , b , c , d , within a treatment indicates significant differences (p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Related Articles

Centrifugation:

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: Then, added 500 μl RIPA lysis buffer and incubated at 4 °C for five min. After that, cells were scraped rapidly with a cells scrapper to remove and lyse residual cells and transferred cell lysate to a tube for centrifugation at 4 °C for 10 min. .. According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Article Title: Centipede grass exerts anti-adipogenic activity through inhibition of C/EBP?, C/EBP?, and PPAR? expression and the AKT signaling pathway in 3T3-L1 adipocytes
Article Snippet: After cell debris was removed by centrifugation, lysate protein concentrations were determined using Bio-Rad protein Assay Reagent (Bio-Rad Laboratories, USA). .. PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from cell signaling and the monoclonal β-actin antibody was from Chemicon.

Article Title: Erigeron annuus (L.) Pers. Extract Inhibits Reactive Oxygen Species (ROS) Production and Fat Accumulation in 3T3-L1 Cells by Activating an AMP-Dependent Kinase Signaling Pathway
Article Snippet: Cells lysates were clarified by centrifugation at 12,000 g for 30 min. .. PPARγ, C/EBPα, SREBP-1c, phospho-AMPK (p -AMPK), total-AMPK, and phospho-ACC (p -ACC), CPT1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Amplification:

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: .. The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. For staining for cardiac troponin T (cTnT), sections were incubated in mouse on mouse diluent working solution for 5 min (Vector Laboratories, M.O.M.

Mass Spectrometry:

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. Basic Kit, BMK-2202) and in mouse monoclonal anti-cTnT (1:200, Thermo Fisher Scientific, MS-295-P1) diluted in M.O.M. diluent for 30 min. After washing, the sections were incubated in Alexa Fluor 546 donkey anti-mouse IgG (1:500, Life Technologies, ) for 1 h. The slides were counterstained with DAPI and mounted with Vectashield hard-set mounting medium (Vector Laboratories).

Nucleic Acid Electrophoresis:

Article Title: Annual Wormwood Leaf Inhibits the Adipogenesis of 3T3-L1 and Obesity in High-Fat Diet-Induced Obese Rats
Article Snippet: Cell lysates (50 μg protein) were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia, Little Chalfont, England, UK), blocked with 5% skim milk and hybridized with primary antibodies. .. PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from Cell Signaling (Danvers, MA, USA) and the monoclonal β-actin antibody was from Chemicon (Temecula, California, USA).

Pyrolysis Gas Chromatography:

Article Title: Antiobesity Effect of Astilbe chinensis Franch. et Savet. Extract through Regulation of Adipogenesis and AMP-Activated Protein Kinase Pathways in 3T3-L1 Adipocyte and High-Fat Diet-Induced C57BL/6N Obese Mice
Article Snippet: .. The primary antibodies PPAR γ , C/EBP α , SREBP1, FAS, SCD-1, phosphor-AMPK (p-AMPK), AMPK, phosphor-ACC (p-ACC), ACC, PGC-1 α , PPAR α , ATGL, phosphor-hormone sensitive lipase (p-HSL), and HSL were purchased form Cell Signaling (Danver, USA). ..

Blocking Assay:

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: .. The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. For staining for cardiac troponin T (cTnT), sections were incubated in mouse on mouse diluent working solution for 5 min (Vector Laboratories, M.O.M.

Electrophoresis:

Article Title: Centipede grass exerts anti-adipogenic activity through inhibition of C/EBP?, C/EBP?, and PPAR? expression and the AKT signaling pathway in 3T3-L1 adipocytes
Article Snippet: Cell lysates were then subjected to electrophoresis on 10-15% SDS-polyacrylamide gels containing SDS, and transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia, UK). .. PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from cell signaling and the monoclonal β-actin antibody was from Chemicon.

Incubation:

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2
Article Snippet: .. According to western breeze chemilumeniscence protocol (Invitrogen), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, and adiponectin, FAS, SREBP-1 and GAPDH. (Cell Signaling Technology, Danvers, MA, USA). .. The signals were observed with an enhanced chemilumeniscence kit (Bio-Rad-USA) by a chemiluminescence imaging system (Davinch Chemi, Seoul, Korea) and the intensity of immune reacted bands was quantified by the ImageJ software (1.49 version(32 bit), Wayne Rasband, National Institute of Health, Bethesda, MD, USA).

Article Title: Annual Wormwood Leaf Inhibits the Adipogenesis of 3T3-L1 and Obesity in High-Fat Diet-Induced Obese Rats
Article Snippet: PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from Cell Signaling (Danvers, MA, USA) and the monoclonal β-actin antibody was from Chemicon (Temecula, California, USA). .. After incubation with horseradish-peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent ECL Assay Kit (Amersham Pharmacia, Little Chalfont, England, UK) according to the manufacturer’s instructions.

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: .. According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA). .. The signals were observed with an enhanced chemiluminescence kit (Biorad, Seoul, South Korea) by a chemiluminescence imaging system (Davinch Chemiluminescence, Seoul, South Korea) and the intensity of immune reacted bands was quantified by the ImageJ software-1.49 version (32 bit), Wayne Rasband, National Institute of Health, USA.

Article Title: Centipede grass exerts anti-adipogenic activity through inhibition of C/EBP?, C/EBP?, and PPAR? expression and the AKT signaling pathway in 3T3-L1 adipocytes
Article Snippet: PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from cell signaling and the monoclonal β-actin antibody was from Chemicon. .. After incubation with horseradish-peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent ECL assay kit (Amersham Pharmacia, UK) according to the manufacturer’s instructions.

Article Title: MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis
Article Snippet: The membrane was rinsed with TBS-Tween20 (TBST), blocked for 2 h in TBST containing 5% (w/v) skimmed milk, and incubated with primary antibody for 1 h. The membrane was washed with TBST and incubated for 1 h with secondary antibody conjugated to HRP. .. The primary antibody for C/EBPα was rabbit anti-C/EBPα (#2295, Cell Signaling Technology), and the secondary antibody was HRP-conjugated goat anti-rabbit IgG.

Article Title: Erigeron annuus (L.) Pers. Extract Inhibits Reactive Oxygen Species (ROS) Production and Fat Accumulation in 3T3-L1 Cells by Activating an AMP-Dependent Kinase Signaling Pathway
Article Snippet: Then 20 μg of the protein extract was mixed with 2 × sample buffer, heated at 95 °C for 5 min, separated by 10% SDS-PAGE, and transferred to PVDF membrane at 100 V for 90 min. Membranes were blocked with 1 × TBST comprising 5% skim milk at room temperature for 1 h, incubated with primary antibody overnight at 4 °C, washed five times with 1 × TBST (10 min each wash), incubated with secondary antibody at room temperature for 1 h, and washed five times with 1 × TBST (10 min each wash). .. PPARγ, C/EBPα, SREBP-1c, phospho-AMPK (p -AMPK), total-AMPK, and phospho-ACC (p -ACC), CPT1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: .. The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. For staining for cardiac troponin T (cTnT), sections were incubated in mouse on mouse diluent working solution for 5 min (Vector Laboratories, M.O.M.

Expressing:

Article Title: miR-148a is Associated with Obesity and Modulates Adipocyte Differentiation of Mesenchymal Stem Cells through Wnt Signaling
Article Snippet: Membranes were probed with primary antibodies against PPARγ2 (Cat. No. 2435S, Cell Signaling Technology, Danvers, MA) and C/EBP-α (Cat. No. 8178S, Cell Signaling Technology, Danvers, MA), Wnt1 (Cat. No. 365800 Invitrogen, Carlsbad, CA), β-catenin (Cat. No. 365800, Invitrogen, Carlsbad, CA), phosphorylated GSK-3β (Cat. No. 5558, Cell Signaling Technology, Danvers, MA), and GSK-3β (Cat. No. 9832S, Cell Signaling Technology, Danvers, MA). .. Membranes were reprobed with anti-GAPDH antibody (Sigma, St. Louis, MO) to normalize expression.

Article Title: Matrix metalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis
Article Snippet: Anti-phospho-Erk1/2, P38, NFkB, C/EBPα, Stat1, and Stat3 were purchased from Cell Signaling Technology. .. Anti-MMP12 Ab was used in combination with the above lineage markers to measure MMP12 protein expression in lal −/− myeloid lineage progenitor cells.

BIA-KA:

Article Title: miR-148a is Associated with Obesity and Modulates Adipocyte Differentiation of Mesenchymal Stem Cells through Wnt Signaling
Article Snippet: Protein concentrations were determined by the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). .. Membranes were probed with primary antibodies against PPARγ2 (Cat. No. 2435S, Cell Signaling Technology, Danvers, MA) and C/EBP-α (Cat. No. 8178S, Cell Signaling Technology, Danvers, MA), Wnt1 (Cat. No. 365800 Invitrogen, Carlsbad, CA), β-catenin (Cat. No. 365800, Invitrogen, Carlsbad, CA), phosphorylated GSK-3β (Cat. No. 5558, Cell Signaling Technology, Danvers, MA), and GSK-3β (Cat. No. 9832S, Cell Signaling Technology, Danvers, MA).

Article Title: Erigeron annuus (L.) Pers. Extract Inhibits Reactive Oxygen Species (ROS) Production and Fat Accumulation in 3T3-L1 Cells by Activating an AMP-Dependent Kinase Signaling Pathway
Article Snippet: Protein concentrations were measured with bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Waltham, MA, USA). .. PPARγ, C/EBPα, SREBP-1c, phospho-AMPK (p -AMPK), total-AMPK, and phospho-ACC (p -ACC), CPT1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Modification:

Article Title: Inhibitory effects of Orostachys malacophyllus var. iwarenge extracts on reactive oxygen species production and lipid accumulation during 3T3-L1 adipocyte differentiation
Article Snippet: Fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM) and antibiotic solution (100X, 100unit) were purchased from Welgene Inc. (Daegu, Korea). .. Anti-NOX4, G6PDH, Cu/Zn-SOD, Mn-SOD, PPARγ, C/EBPα, aP2, β-actin and horseradish peroxidase (HRP)-conjugated anti-rabbit were obtained from Cell Signaling Technology (Danvers, MA, USA).

Western Blot:

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2
Article Snippet: .. According to western breeze chemilumeniscence protocol (Invitrogen), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, and adiponectin, FAS, SREBP-1 and GAPDH. (Cell Signaling Technology, Danvers, MA, USA). .. The signals were observed with an enhanced chemilumeniscence kit (Bio-Rad-USA) by a chemiluminescence imaging system (Davinch Chemi, Seoul, Korea) and the intensity of immune reacted bands was quantified by the ImageJ software (1.49 version(32 bit), Wayne Rasband, National Institute of Health, Bethesda, MD, USA).

Article Title: Antiobesity Effect of Astilbe chinensis Franch. et Savet. Extract through Regulation of Adipogenesis and AMP-Activated Protein Kinase Pathways in 3T3-L1 Adipocyte and High-Fat Diet-Induced C57BL/6N Obese Mice
Article Snippet: Paragraph title: 2.9. Western Blot Analysis ... The primary antibodies PPAR γ , C/EBP α , SREBP1, FAS, SCD-1, phosphor-AMPK (p-AMPK), AMPK, phosphor-ACC (p-ACC), ACC, PGC-1 α , PPAR α , ATGL, phosphor-hormone sensitive lipase (p-HSL), and HSL were purchased form Cell Signaling (Danver, USA).

Article Title: Annual Wormwood Leaf Inhibits the Adipogenesis of 3T3-L1 and Obesity in High-Fat Diet-Induced Obese Rats
Article Snippet: Paragraph title: 2.6. Western Blot Analysis ... PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from Cell Signaling (Danvers, MA, USA) and the monoclonal β-actin antibody was from Chemicon (Temecula, California, USA).

Article Title: MORC2 regulates C/EBPα-mediated cell differentiation via sumoylation
Article Snippet: Paragraph title: GST pull-down assay, Western blot, and immunoprecipitation assay ... Primary antibody dilution and order company of His (1:2000, GenScript Corporation), c-myc and sumo1 (1:1000, Santa Cruz) and C/EBPα and Ubiquitin(1:1000, Cell Signaling Technology); Flag (1:2000, Shang Hai, Genomics); MORC2 (1:2000, Abcam);TFF1(1:1000, Protein tech Group).

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: .. According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA). .. The signals were observed with an enhanced chemiluminescence kit (Biorad, Seoul, South Korea) by a chemiluminescence imaging system (Davinch Chemiluminescence, Seoul, South Korea) and the intensity of immune reacted bands was quantified by the ImageJ software-1.49 version (32 bit), Wayne Rasband, National Institute of Health, USA.

Article Title: Centipede grass exerts anti-adipogenic activity through inhibition of C/EBP?, C/EBP?, and PPAR? expression and the AKT signaling pathway in 3T3-L1 adipocytes
Article Snippet: Paragraph title: Western blot analysis ... PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from cell signaling and the monoclonal β-actin antibody was from Chemicon.

Article Title: miR-148a is Associated with Obesity and Modulates Adipocyte Differentiation of Mesenchymal Stem Cells through Wnt Signaling
Article Snippet: Paragraph title: Western blot ... Membranes were probed with primary antibodies against PPARγ2 (Cat. No. 2435S, Cell Signaling Technology, Danvers, MA) and C/EBP-α (Cat. No. 8178S, Cell Signaling Technology, Danvers, MA), Wnt1 (Cat. No. 365800 Invitrogen, Carlsbad, CA), β-catenin (Cat. No. 365800, Invitrogen, Carlsbad, CA), phosphorylated GSK-3β (Cat. No. 5558, Cell Signaling Technology, Danvers, MA), and GSK-3β (Cat. No. 9832S, Cell Signaling Technology, Danvers, MA).

Article Title: MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis
Article Snippet: Paragraph title: Western Blotting ... The primary antibody for C/EBPα was rabbit anti-C/EBPα (#2295, Cell Signaling Technology), and the secondary antibody was HRP-conjugated goat anti-rabbit IgG.

Article Title: Erigeron annuus (L.) Pers. Extract Inhibits Reactive Oxygen Species (ROS) Production and Fat Accumulation in 3T3-L1 Cells by Activating an AMP-Dependent Kinase Signaling Pathway
Article Snippet: PPARγ, C/EBPα, SREBP-1c, phospho-AMPK (p -AMPK), total-AMPK, and phospho-ACC (p -ACC), CPT1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). .. The target protein was detected with Luminata ™ Forte Western HRP substrate (Millipore, Tokyo, Japan).

Protease Inhibitor:

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2
Article Snippet: Western Blot Proteins were extracted from differentiated adipocytes using RIPA lysis buffer with 1× protease inhibitor cocktail (Roche, Basel, Switzerland). .. According to western breeze chemilumeniscence protocol (Invitrogen), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, and adiponectin, FAS, SREBP-1 and GAPDH. (Cell Signaling Technology, Danvers, MA, USA).

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: Protein extraction and immune blot analysis Proteins from experimental adipocyte were extracted by RIPA lysis buffer with 1X protease inhibitor cocktail (Roche, Basel, Switzerland). .. According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Article Title: miR-148a is Associated with Obesity and Modulates Adipocyte Differentiation of Mesenchymal Stem Cells through Wnt Signaling
Article Snippet: Western blot hMSCs-Ad were lysed in immunoprecipitation (IP) assay buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) containing a protease inhibitor mixture (Roche Applied Science, Penzberg, Upper Bavaria, Germany). .. Membranes were probed with primary antibodies against PPARγ2 (Cat. No. 2435S, Cell Signaling Technology, Danvers, MA) and C/EBP-α (Cat. No. 8178S, Cell Signaling Technology, Danvers, MA), Wnt1 (Cat. No. 365800 Invitrogen, Carlsbad, CA), β-catenin (Cat. No. 365800, Invitrogen, Carlsbad, CA), phosphorylated GSK-3β (Cat. No. 5558, Cell Signaling Technology, Danvers, MA), and GSK-3β (Cat. No. 9832S, Cell Signaling Technology, Danvers, MA).

Imaging:

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2
Article Snippet: According to western breeze chemilumeniscence protocol (Invitrogen), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, and adiponectin, FAS, SREBP-1 and GAPDH. (Cell Signaling Technology, Danvers, MA, USA). .. The signals were observed with an enhanced chemilumeniscence kit (Bio-Rad-USA) by a chemiluminescence imaging system (Davinch Chemi, Seoul, Korea) and the intensity of immune reacted bands was quantified by the ImageJ software (1.49 version(32 bit), Wayne Rasband, National Institute of Health, Bethesda, MD, USA).

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA). .. The signals were observed with an enhanced chemiluminescence kit (Biorad, Seoul, South Korea) by a chemiluminescence imaging system (Davinch Chemiluminescence, Seoul, South Korea) and the intensity of immune reacted bands was quantified by the ImageJ software-1.49 version (32 bit), Wayne Rasband, National Institute of Health, USA.

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. A Zeiss LSM 780 confocal microscope was used for imaging.

Protein Concentration:

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2
Article Snippet: The total protein concentration was measured by a Bio-Rad protein assay kit. .. According to western breeze chemilumeniscence protocol (Invitrogen), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, and adiponectin, FAS, SREBP-1 and GAPDH. (Cell Signaling Technology, Danvers, MA, USA).

Article Title: Antiobesity Effect of Astilbe chinensis Franch. et Savet. Extract through Regulation of Adipogenesis and AMP-Activated Protein Kinase Pathways in 3T3-L1 Adipocyte and High-Fat Diet-Induced C57BL/6N Obese Mice
Article Snippet: The protein concentration of the cell lysates was measured using a Bio-Rad protein assay kit (Hercules, CA). .. The primary antibodies PPAR γ , C/EBP α , SREBP1, FAS, SCD-1, phosphor-AMPK (p-AMPK), AMPK, phosphor-ACC (p-ACC), ACC, PGC-1 α , PPAR α , ATGL, phosphor-hormone sensitive lipase (p-HSL), and HSL were purchased form Cell Signaling (Danver, USA).

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: The total protein concentration was measured by a Bio-Rad protein assay kit. .. According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Article Title: MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis
Article Snippet: Western Blotting Cells at different days after MDI stimulation were lysed in cell lysis buffer (Beyotime) containing 1 mM phenylmethylsulfonyl fluoride, and the protein concentration was quantified as mentioned above. .. The primary antibody for C/EBPα was rabbit anti-C/EBPα (#2295, Cell Signaling Technology), and the secondary antibody was HRP-conjugated goat anti-rabbit IgG.

Immunofluorescence:

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: Paragraph title: Immunofluorescence ... The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT).

MTT Assay:

Article Title: Inhibitory effects of Orostachys malacophyllus var. iwarenge extracts on reactive oxygen species production and lipid accumulation during 3T3-L1 adipocyte differentiation
Article Snippet: Gallic acid, Folin-Ciocalteu phenol reagent, ascorbic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulfate, sodium phosphate, sodium carbonate, isobutylmethyl-xanthine (IBMX), insulin, dexamethasone (DEX), dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium-bromide (MTT), dimethyl sulfoxide (DMSO), isopropanol, N -acetyl- l -cysteine (NAC), nitroblue tetrazolium (NBT), fluorescein sodium salt (FL) and Oil Red O were purchased from Sigma (St. Louis, MO, USA). .. Anti-NOX4, G6PDH, Cu/Zn-SOD, Mn-SOD, PPARγ, C/EBPα, aP2, β-actin and horseradish peroxidase (HRP)-conjugated anti-rabbit were obtained from Cell Signaling Technology (Danvers, MA, USA).

Pull Down Assay:

Article Title: MORC2 regulates C/EBPα-mediated cell differentiation via sumoylation
Article Snippet: Paragraph title: GST pull-down assay, Western blot, and immunoprecipitation assay ... Primary antibody dilution and order company of His (1:2000, GenScript Corporation), c-myc and sumo1 (1:1000, Santa Cruz) and C/EBPα and Ubiquitin(1:1000, Cell Signaling Technology); Flag (1:2000, Shang Hai, Genomics); MORC2 (1:2000, Abcam);TFF1(1:1000, Protein tech Group).

Fluorescence:

Article Title: Matrix metalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis
Article Snippet: Anti-phospho-Erk1/2, P38, NFkB, C/EBPα, Stat1, and Stat3 were purchased from Cell Signaling Technology. .. Percentage cell numbers and mean fluorescence intensity (MFI) were analyzed using the BD FACStation Software (BD Biosciences).

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: .. The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. For staining for cardiac troponin T (cTnT), sections were incubated in mouse on mouse diluent working solution for 5 min (Vector Laboratories, M.O.M.

Microscopy:

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. A Zeiss LSM 780 confocal microscope was used for imaging.

Protein Extraction:

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: Paragraph title: Protein extraction and immune blot analysis ... According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA).

FACS:

Article Title: Matrix metalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis
Article Snippet: Paragraph title: FACS analysis ... Anti-phospho-Erk1/2, P38, NFkB, C/EBPα, Stat1, and Stat3 were purchased from Cell Signaling Technology.

Lysis:

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2
Article Snippet: Western Blot Proteins were extracted from differentiated adipocytes using RIPA lysis buffer with 1× protease inhibitor cocktail (Roche, Basel, Switzerland). .. According to western breeze chemilumeniscence protocol (Invitrogen), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, and adiponectin, FAS, SREBP-1 and GAPDH. (Cell Signaling Technology, Danvers, MA, USA).

Article Title: Antiobesity Effect of Astilbe chinensis Franch. et Savet. Extract through Regulation of Adipogenesis and AMP-Activated Protein Kinase Pathways in 3T3-L1 Adipocyte and High-Fat Diet-Induced C57BL/6N Obese Mice
Article Snippet: To detect proteins from whole adipocyte cell lysates, 3T3-L1 cells and epididymal adipose tissue were homogenized in lysis buffer. .. The primary antibodies PPAR γ , C/EBP α , SREBP1, FAS, SCD-1, phosphor-AMPK (p-AMPK), AMPK, phosphor-ACC (p-ACC), ACC, PGC-1 α , PPAR α , ATGL, phosphor-hormone sensitive lipase (p-HSL), and HSL were purchased form Cell Signaling (Danver, USA).

Article Title: Annual Wormwood Leaf Inhibits the Adipogenesis of 3T3-L1 and Obesity in High-Fat Diet-Induced Obese Rats
Article Snippet: Briefly, cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2 , 0.1% sodium dodecyl sulfate (SDS), 2 mM phenylmethylsulfonyl fluoride, 80 μg/mL leupeptin, 3 mM NaF and 1 mM Dithiothreitol (DTT). .. PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from Cell Signaling (Danvers, MA, USA) and the monoclonal β-actin antibody was from Chemicon (Temecula, California, USA).

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: Then, added 500 μl RIPA lysis buffer and incubated at 4 °C for five min. After that, cells were scraped rapidly with a cells scrapper to remove and lyse residual cells and transferred cell lysate to a tube for centrifugation at 4 °C for 10 min. .. According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Article Title: Centipede grass exerts anti-adipogenic activity through inhibition of C/EBP?, C/EBP?, and PPAR? expression and the AKT signaling pathway in 3T3-L1 adipocytes
Article Snippet: Briefly, cells were washed with ice-cold PBS, and lysed in lysis buffer (50 mM Tris–HCl (pH 8.0), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2 , 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 80 μg/ml leupeptin, 3 mM NaF and 1 mM DTT). .. PPARγ, C/EBPβ, C/EBPα, aP2, Akt, and GSK3β antibody were from cell signaling and the monoclonal β-actin antibody was from Chemicon.

Article Title: MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis
Article Snippet: Western Blotting Cells at different days after MDI stimulation were lysed in cell lysis buffer (Beyotime) containing 1 mM phenylmethylsulfonyl fluoride, and the protein concentration was quantified as mentioned above. .. The primary antibody for C/EBPα was rabbit anti-C/EBPα (#2295, Cell Signaling Technology), and the secondary antibody was HRP-conjugated goat anti-rabbit IgG.

SDS Page:

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2
Article Snippet: An equal amount of protein samples were separated by SDS-PAGE (10%) and transblotted onto polyvinylidene difluoride (PVDF) membranes (iBlot gel transfer stacks, Novex, Life Technology, Waltham, MA, USA). .. According to western breeze chemilumeniscence protocol (Invitrogen), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, and adiponectin, FAS, SREBP-1 and GAPDH. (Cell Signaling Technology, Danvers, MA, USA).

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: An equal amount of protein samples were separated by SDS-PAGE (10 %) and trans blotted onto polyvinylidene difluoride (PVDF) membranes (iBlot gel transfer stacks, Novex, Life Technology, Waltham, MA, USA). .. According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Article Title: miR-148a is Associated with Obesity and Modulates Adipocyte Differentiation of Mesenchymal Stem Cells through Wnt Signaling
Article Snippet: Proteins were separated on a 10% SDS/PAGE gel under reduction conditions and electroblotted onto a PVDF membrane. .. Membranes were probed with primary antibodies against PPARγ2 (Cat. No. 2435S, Cell Signaling Technology, Danvers, MA) and C/EBP-α (Cat. No. 8178S, Cell Signaling Technology, Danvers, MA), Wnt1 (Cat. No. 365800 Invitrogen, Carlsbad, CA), β-catenin (Cat. No. 365800, Invitrogen, Carlsbad, CA), phosphorylated GSK-3β (Cat. No. 5558, Cell Signaling Technology, Danvers, MA), and GSK-3β (Cat. No. 9832S, Cell Signaling Technology, Danvers, MA).

Article Title: MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis
Article Snippet: Equal amounts of total cellular protein were fractionated by 12% (w/v) SDS/PAGE and electronically transferred to 0.2 µm PVDF membrane (Bio-Rad). .. The primary antibody for C/EBPα was rabbit anti-C/EBPα (#2295, Cell Signaling Technology), and the secondary antibody was HRP-conjugated goat anti-rabbit IgG.

Article Title: Erigeron annuus (L.) Pers. Extract Inhibits Reactive Oxygen Species (ROS) Production and Fat Accumulation in 3T3-L1 Cells by Activating an AMP-Dependent Kinase Signaling Pathway
Article Snippet: Then 20 μg of the protein extract was mixed with 2 × sample buffer, heated at 95 °C for 5 min, separated by 10% SDS-PAGE, and transferred to PVDF membrane at 100 V for 90 min. Membranes were blocked with 1 × TBST comprising 5% skim milk at room temperature for 1 h, incubated with primary antibody overnight at 4 °C, washed five times with 1 × TBST (10 min each wash), incubated with secondary antibody at room temperature for 1 h, and washed five times with 1 × TBST (10 min each wash). .. PPARγ, C/EBPα, SREBP-1c, phospho-AMPK (p -AMPK), total-AMPK, and phospho-ACC (p -ACC), CPT1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Plasmid Preparation:

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT).

Software:

Article Title: Phenyllactic Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-γ2
Article Snippet: According to western breeze chemilumeniscence protocol (Invitrogen), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, and adiponectin, FAS, SREBP-1 and GAPDH. (Cell Signaling Technology, Danvers, MA, USA). .. The signals were observed with an enhanced chemilumeniscence kit (Bio-Rad-USA) by a chemiluminescence imaging system (Davinch Chemi, Seoul, Korea) and the intensity of immune reacted bands was quantified by the ImageJ software (1.49 version(32 bit), Wayne Rasband, National Institute of Health, Bethesda, MD, USA).

Article Title: Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach
Article Snippet: According to western breeze chemiluminescence protocol (Invitrogen, USA), the immunobloting was performed (except primary antibody incubation time and temperature) with rabbit monoclonal antibody of specific proteins such as PPRAγ2, C/EBP-α, adiponectin, AMPK-α and β-actin (Cell Signaling Technology, Danvers, MA, USA). .. The signals were observed with an enhanced chemiluminescence kit (Biorad, Seoul, South Korea) by a chemiluminescence imaging system (Davinch Chemiluminescence, Seoul, South Korea) and the intensity of immune reacted bands was quantified by the ImageJ software-1.49 version (32 bit), Wayne Rasband, National Institute of Health, USA.

Article Title: Matrix metalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis
Article Snippet: Anti-phospho-Erk1/2, P38, NFkB, C/EBPα, Stat1, and Stat3 were purchased from Cell Signaling Technology. .. Percentage cell numbers and mean fluorescence intensity (MFI) were analyzed using the BD FACStation Software (BD Biosciences).

In Vitro:

Article Title: MORC2 regulates C/EBPα-mediated cell differentiation via sumoylation
Article Snippet: GST pull-down assay, Western blot, and immunoprecipitation assay In vitro transcription and translation of His-MORC2 or Flag-C/EBPα proteins were performed by using the TNT-coupled transcription and translation system (Promega), as previously described [ ]. .. Primary antibody dilution and order company of His (1:2000, GenScript Corporation), c-myc and sumo1 (1:1000, Santa Cruz) and C/EBPα and Ubiquitin(1:1000, Cell Signaling Technology); Flag (1:2000, Shang Hai, Genomics); MORC2 (1:2000, Abcam);TFF1(1:1000, Protein tech Group).

Immunoprecipitation:

Article Title: MORC2 regulates C/EBPα-mediated cell differentiation via sumoylation
Article Snippet: Paragraph title: GST pull-down assay, Western blot, and immunoprecipitation assay ... Primary antibody dilution and order company of His (1:2000, GenScript Corporation), c-myc and sumo1 (1:1000, Santa Cruz) and C/EBPα and Ubiquitin(1:1000, Cell Signaling Technology); Flag (1:2000, Shang Hai, Genomics); MORC2 (1:2000, Abcam);TFF1(1:1000, Protein tech Group).

Article Title: miR-148a is Associated with Obesity and Modulates Adipocyte Differentiation of Mesenchymal Stem Cells through Wnt Signaling
Article Snippet: Western blot hMSCs-Ad were lysed in immunoprecipitation (IP) assay buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) containing a protease inhibitor mixture (Roche Applied Science, Penzberg, Upper Bavaria, Germany). .. Membranes were probed with primary antibodies against PPARγ2 (Cat. No. 2435S, Cell Signaling Technology, Danvers, MA) and C/EBP-α (Cat. No. 8178S, Cell Signaling Technology, Danvers, MA), Wnt1 (Cat. No. 365800 Invitrogen, Carlsbad, CA), β-catenin (Cat. No. 365800, Invitrogen, Carlsbad, CA), phosphorylated GSK-3β (Cat. No. 5558, Cell Signaling Technology, Danvers, MA), and GSK-3β (Cat. No. 9832S, Cell Signaling Technology, Danvers, MA).

Marker:

Article Title: Matrix metalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis
Article Snippet: Lineage markers (biotin-CD3, -CD4, -CD8, -Mac-1, -Gr-1, -Ter119, and -B220) and other marker Abs (Sca-1, c-Kit, IL7Rα, and CD34) were purchased from BD Biosciences. .. Anti-phospho-Erk1/2, P38, NFkB, C/EBPα, Stat1, and Stat3 were purchased from Cell Signaling Technology.

Staining:

Article Title: Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition
Article Snippet: .. The tissue sections were treated with 0.05% trypsin (HyClone, SH30042.01) for 20 min at 37°C, 0.1% Tween-20 in PBS for 5 min, 0.5% Tween-20 in PBS for 5 min, 0.5% Triton X-100 in PBS for 15 min and 0.3% H2 O2 in PBS for 15 min, and were then blocked with 10% donkey serum in PBS with 0.1% Tween-20 for 1 h. For staining of C/EBPα, sections were incubated with rabbit monoclonal anti-C/EBPα antibody (1:200, Cell Signaling Technology, 8178) in blocking buffer overnight at 4°C, washed and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200, Vector Laboratories, PI-1000) for 30 min. After washing, sections were incubated in tyramide signal amplification working solution for 10 min (1:100, PerkinElmer, TSA Plus Fluorescence kits, NEL741001KT). .. For staining for cardiac troponin T (cTnT), sections were incubated in mouse on mouse diluent working solution for 5 min (Vector Laboratories, M.O.M.

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    Cell Signaling Technology Inc rabbit ps21 c ebpα
    MV4;11 cells differentiate upon inhibition of the ERK1/2 pathway. (A) Western blot showing decreased <t>pS21-C/EBPα</t> (top) in response to continuous treatment with 100 μM of the MEK1 inhibitor PD98059 or vehicle control (DMSO). For DMSO-treated cells, a 72-h incubation is shown. (bottom) The same blot stained with C/EBPα antibody. (B) (top) MV4;11 cell differentiation was monitored by morphological examination of Wright-Giemsa stained cytospin preparations. Red arrows point to cells with granulocytic morphology. (bottom) Respiratory burst activity as assessed by the NBT assay.
    Rabbit Ps21 C Ebpα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit ps21 c ebpα/product/Cell Signaling Technology Inc
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit ps21 c ebpα - by Bioz Stars, 2020-04
    85/100 stars
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    99
    Cell Signaling Technology Inc c ebp α
    Combination treatment triggers myeloid differentiation of leukemic cells. FLT3-ITD mutated cell lines MOLM13 (A), MOLM14 (B) and primary AML patients’ samples (C) were treated with the indicated concentrations of selinexor and/or sorafenib for 5 or 6 days in vitro ; morphological changes of the cells were checked with Giemsa staining. Expression of the myeloid differentiation marker CD11b was determined using flow cytometry. The histograms present individual assays which measured fluorescent intensity of CD11b. Each column was generated from four individual assays. (D) Differentiation-related proteins were evaluated using immunoblotting. The expression levels and ratios of <t>C/EBPα/PU-1</t> were measured (first lane which was defined as 1). Dimethylsulfoxide (DMSO) was used as a control. Seli: selinexor; Sora: sorafenib; Combi: combination.
    C Ebp α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c ebp α/product/Cell Signaling Technology Inc
    Average 99 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    c ebp α - by Bioz Stars, 2020-04
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    Image Search Results


    MV4;11 cells differentiate upon inhibition of the ERK1/2 pathway. (A) Western blot showing decreased pS21-C/EBPα (top) in response to continuous treatment with 100 μM of the MEK1 inhibitor PD98059 or vehicle control (DMSO). For DMSO-treated cells, a 72-h incubation is shown. (bottom) The same blot stained with C/EBPα antibody. (B) (top) MV4;11 cell differentiation was monitored by morphological examination of Wright-Giemsa stained cytospin preparations. Red arrows point to cells with granulocytic morphology. (bottom) Respiratory burst activity as assessed by the NBT assay.

    Journal: The Journal of Experimental Medicine

    Article Title: Block of C/EBP? function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations

    doi: 10.1084/jem.20052242

    Figure Lengend Snippet: MV4;11 cells differentiate upon inhibition of the ERK1/2 pathway. (A) Western blot showing decreased pS21-C/EBPα (top) in response to continuous treatment with 100 μM of the MEK1 inhibitor PD98059 or vehicle control (DMSO). For DMSO-treated cells, a 72-h incubation is shown. (bottom) The same blot stained with C/EBPα antibody. (B) (top) MV4;11 cell differentiation was monitored by morphological examination of Wright-Giemsa stained cytospin preparations. Red arrows point to cells with granulocytic morphology. (bottom) Respiratory burst activity as assessed by the NBT assay.

    Article Snippet: The primary antibodies were rabbit pS21-C/EBPα (1:1,000; Cell Signaling), goat C/EBPα (1:1,000; Santa Cruz Biotechnology, Inc.), pY591-FLT3 (1:1,000 54H1; Cell Signaling), rabbit p(T202/Y204)-ERK1/2 (1:1,000; Cell Signaling), and β-tubulin (1:1,000 clone 2–28-33; Sigma-Aldrich).

    Techniques: Inhibition, Western Blot, Incubation, Staining, Cell Differentiation, Activity Assay

    Activation of FLT3 in human AML cells leads to C/EBPα protein phosphorylation on S21 by the ERK1/2 pathway. (A) COS7 cells were untreated or treated with UV-C (60 J/m2; UV) or 100 nM PMA and incubated for 60 min. Endogenous c-Jun NH 2 -terminal protein kinase (JNK), p38, and ERK were immunoprecipitated and used for in vitro kinase assays. Control tests showed that ERK phosphorylated Elk1, JNK-phosphorylated c-Jun, and p38 MAP kinase phosphorylated ATF2. (B) THP-1 cells expressing wild-type FLT3 were serum starved for 6 h (0), or starved and stimulated with 100 ng/ml FLT3 ligand (FL) for 5 and 10 min before harvest. Western blot of whole cell extracts was sequentially stained with pS21-C/EBPα, C/EBPα, pThr202/Tyr204-ERK1/2, and β-tubulin antibodies. (C) Human AML cell lines with mutant FLT3 have increased phosphorylation of C/EBPα on S21. All cell lines were serum starved for 7 h in the absence (−) or presence (+) of 1 μM MLN518. Western blot was analyzed as in B. (D) MV4;11 cells were serum starved in the absence (−) or presence of the FLT3 inhibitors AG1296 and MLN518 at concentrations marked above the lanes, or with vehicle control (DMSO). Western blot analyses were done as in B. (E) Phosphorylation status of C/EBPα in MV4;11 and MOLM-13 cells as a dose response to MLN518. Cells were serum starved for 7 h in the presence of MLN518 at concentrations indicated above the lanes. Western blot was stained as in B. (F) Time course of FLT3 inhibition and its effect on C/EBPα phosphorylation. At each time point, MV4;11, MOLM-13, or MOLM-14 cells were serum starved for a total period of 8 h. MLN518 (1 μM) was added for the final hours (indicated above the lanes) of the treatment. For example, “1 hr” means that the cells were starved for 7 h in the absence of the inhibitor, and for 1 final hour in its presence. Western blot staining was as described previously. (G) Decrease of pS21-C/EBPα by inhibition of FLT3 activity in AML patient samples. Leukemic blasts from bone marrow (patient no. 592), or peripheral blood (patient no. 667) were cultured in the absence (0), or presence ( 1 ) of MLN518 for 6 h. Western blot of whole cell extracts was analyzed with antibodies recognizing activated FLT3 (P-FLT3), phosphorylated C/EBPα (P-Ser21-C/EBPα), or total C/EBPα protein. MV4;11 cells were included for control. Quantification of C/EBPα phosphorylation normalized to the total C/EBPα protein is shown (bottom).

    Journal: The Journal of Experimental Medicine

    Article Title: Block of C/EBP? function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations

    doi: 10.1084/jem.20052242

    Figure Lengend Snippet: Activation of FLT3 in human AML cells leads to C/EBPα protein phosphorylation on S21 by the ERK1/2 pathway. (A) COS7 cells were untreated or treated with UV-C (60 J/m2; UV) or 100 nM PMA and incubated for 60 min. Endogenous c-Jun NH 2 -terminal protein kinase (JNK), p38, and ERK were immunoprecipitated and used for in vitro kinase assays. Control tests showed that ERK phosphorylated Elk1, JNK-phosphorylated c-Jun, and p38 MAP kinase phosphorylated ATF2. (B) THP-1 cells expressing wild-type FLT3 were serum starved for 6 h (0), or starved and stimulated with 100 ng/ml FLT3 ligand (FL) for 5 and 10 min before harvest. Western blot of whole cell extracts was sequentially stained with pS21-C/EBPα, C/EBPα, pThr202/Tyr204-ERK1/2, and β-tubulin antibodies. (C) Human AML cell lines with mutant FLT3 have increased phosphorylation of C/EBPα on S21. All cell lines were serum starved for 7 h in the absence (−) or presence (+) of 1 μM MLN518. Western blot was analyzed as in B. (D) MV4;11 cells were serum starved in the absence (−) or presence of the FLT3 inhibitors AG1296 and MLN518 at concentrations marked above the lanes, or with vehicle control (DMSO). Western blot analyses were done as in B. (E) Phosphorylation status of C/EBPα in MV4;11 and MOLM-13 cells as a dose response to MLN518. Cells were serum starved for 7 h in the presence of MLN518 at concentrations indicated above the lanes. Western blot was stained as in B. (F) Time course of FLT3 inhibition and its effect on C/EBPα phosphorylation. At each time point, MV4;11, MOLM-13, or MOLM-14 cells were serum starved for a total period of 8 h. MLN518 (1 μM) was added for the final hours (indicated above the lanes) of the treatment. For example, “1 hr” means that the cells were starved for 7 h in the absence of the inhibitor, and for 1 final hour in its presence. Western blot staining was as described previously. (G) Decrease of pS21-C/EBPα by inhibition of FLT3 activity in AML patient samples. Leukemic blasts from bone marrow (patient no. 592), or peripheral blood (patient no. 667) were cultured in the absence (0), or presence ( 1 ) of MLN518 for 6 h. Western blot of whole cell extracts was analyzed with antibodies recognizing activated FLT3 (P-FLT3), phosphorylated C/EBPα (P-Ser21-C/EBPα), or total C/EBPα protein. MV4;11 cells were included for control. Quantification of C/EBPα phosphorylation normalized to the total C/EBPα protein is shown (bottom).

    Article Snippet: The primary antibodies were rabbit pS21-C/EBPα (1:1,000; Cell Signaling), goat C/EBPα (1:1,000; Santa Cruz Biotechnology, Inc.), pY591-FLT3 (1:1,000 54H1; Cell Signaling), rabbit p(T202/Y204)-ERK1/2 (1:1,000; Cell Signaling), and β-tubulin (1:1,000 clone 2–28-33; Sigma-Aldrich).

    Techniques: Activation Assay, Incubation, Immunoprecipitation, In Vitro, Expressing, Western Blot, Staining, Mutagenesis, Inhibition, Activity Assay, Cell Culture

    Combination treatment triggers myeloid differentiation of leukemic cells. FLT3-ITD mutated cell lines MOLM13 (A), MOLM14 (B) and primary AML patients’ samples (C) were treated with the indicated concentrations of selinexor and/or sorafenib for 5 or 6 days in vitro ; morphological changes of the cells were checked with Giemsa staining. Expression of the myeloid differentiation marker CD11b was determined using flow cytometry. The histograms present individual assays which measured fluorescent intensity of CD11b. Each column was generated from four individual assays. (D) Differentiation-related proteins were evaluated using immunoblotting. The expression levels and ratios of C/EBPα/PU-1 were measured (first lane which was defined as 1). Dimethylsulfoxide (DMSO) was used as a control. Seli: selinexor; Sora: sorafenib; Combi: combination.

    Journal: Haematologica

    Article Title: Combinatorial targeting of XPO1 and FLT3 exerts synergistic anti-leukemia effects through induction of differentiation and apoptosis in FLT3-mutated acute myeloid leukemias: from concept to clinical trial

    doi: 10.3324/haematol.2017.185082

    Figure Lengend Snippet: Combination treatment triggers myeloid differentiation of leukemic cells. FLT3-ITD mutated cell lines MOLM13 (A), MOLM14 (B) and primary AML patients’ samples (C) were treated with the indicated concentrations of selinexor and/or sorafenib for 5 or 6 days in vitro ; morphological changes of the cells were checked with Giemsa staining. Expression of the myeloid differentiation marker CD11b was determined using flow cytometry. The histograms present individual assays which measured fluorescent intensity of CD11b. Each column was generated from four individual assays. (D) Differentiation-related proteins were evaluated using immunoblotting. The expression levels and ratios of C/EBPα/PU-1 were measured (first lane which was defined as 1). Dimethylsulfoxide (DMSO) was used as a control. Seli: selinexor; Sora: sorafenib; Combi: combination.

    Article Snippet: The antibodies against human phosphorylated (p)-p44/42 MAPK (ERK1/2)(Thr202/Tyr204), phospho-AKT(Ser473), phospho-FLT3(Tyr589/591), phospho-S6K(Ser240/244), AKT, S6K, Bcl-xL, C/EBPα, PU.1, STAT3, c-Myc and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA), against Bcl-2 from Dako (Carpinteria, CA, USA), against phospho-STAT5 A/B from Upstate (Lake Placid, NY, USA), against total STAT5A/B from R & D Systems Inc. (Minneapolis, MN, USA), against ERK2, FLT3, p53, IκB alpha, phospho-Stat3, and Mcl-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), against Bim and Puma from CalBiochem (San Diego, CA, USA), against HIF1α from BD Biosciences (San Diego, CA, USA), and against phospho-IκB alpha (ser32/36) from Novus (Littleton, CO, USA).

    Techniques: In Vitro, Staining, Expressing, Marker, Flow Cytometry, Cytometry, Generated

    Model for the regulation of CRABP-II expression during adipogenesis and in mature adipocytes. The classical adipocyte differentiation inducers, insulin, IBMX, and dexamethasone ( dex ), all repress the expression of CRABP-II. IBMX increases cellular levels of cAMP leading to activation of PKA. Activated PKA phosphorylates CREB, which positively regulates the expression of its direct target gene CREM. In turn, CREM associates with a CRE in the first intron of CRABP-II and recruits HDAC1 to repress CRABP-II transcription. Dexamethasone activates GR. In turn, GR binds to a GRE in the CRABP-II promoter and negatively regulates CRABP-II expression. The details of the mechanism through which insulin represses CRABP-II expression remain to be explored. In mature adipocytes, low levels of expression of CRABP-II are maintained by direct repression by C/EBPα.

    Journal: The Journal of Biological Chemistry

    Article Title: Repression of Cellular Retinoic Acid-binding Protein II during Adipocyte Differentiation *

    doi: 10.1074/jbc.M110.110635

    Figure Lengend Snippet: Model for the regulation of CRABP-II expression during adipogenesis and in mature adipocytes. The classical adipocyte differentiation inducers, insulin, IBMX, and dexamethasone ( dex ), all repress the expression of CRABP-II. IBMX increases cellular levels of cAMP leading to activation of PKA. Activated PKA phosphorylates CREB, which positively regulates the expression of its direct target gene CREM. In turn, CREM associates with a CRE in the first intron of CRABP-II and recruits HDAC1 to repress CRABP-II transcription. Dexamethasone activates GR. In turn, GR binds to a GRE in the CRABP-II promoter and negatively regulates CRABP-II expression. The details of the mechanism through which insulin represses CRABP-II expression remain to be explored. In mature adipocytes, low levels of expression of CRABP-II are maintained by direct repression by C/EBPα.

    Article Snippet: C/EBPα and CREB antibodies were from Cell Signaling and Millipore, respectively.

    Techniques: Expressing, Activation Assay

    Effects of different doses of metformin (1.25–10 mM) on the expression of adipogenesis markers FASN, PPARγ, and C/EBPα in 3T3-L1 cells. ( A ) Expression levels at day 5 (DAY 5) after differentiation initiation as determined by Western blot analysis ( n = 3); ( B ) Expression levels at day 9 (DAY 9) ( n = 3). Rosiglitazone (Rosi, 2.5 µM) was used as a positive control. Results were quantified using densitometry analysis and normalized to β-Actin. The data represent the means ± SEM from three independent experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Dual Effects of Metformin on Adipogenic Differentiation of 3T3-L1 Preadipocyte in AMPK-Dependent and Independent Manners

    doi: 10.3390/ijms19061547

    Figure Lengend Snippet: Effects of different doses of metformin (1.25–10 mM) on the expression of adipogenesis markers FASN, PPARγ, and C/EBPα in 3T3-L1 cells. ( A ) Expression levels at day 5 (DAY 5) after differentiation initiation as determined by Western blot analysis ( n = 3); ( B ) Expression levels at day 9 (DAY 9) ( n = 3). Rosiglitazone (Rosi, 2.5 µM) was used as a positive control. Results were quantified using densitometry analysis and normalized to β-Actin. The data represent the means ± SEM from three independent experiments. * p

    Article Snippet: Phospho-AMPK, total AMPK, Phospho-ERK, total ERK, phosho-p38, total p38, phospho-JNK, total JNK, phospho-Akt, total Akt, FASN, PPARγ, C/EBPα, polyclonal β-Actin, and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot, Positive Control