adipsin p 16 antibody  (Santa Cruz Biotechnology)

Journal: Endocrinology

Article Title: Inactivation of histone deacetylase 1 (HDAC1) but not HDAC2 is required for the glucocorticoid-dependent CCAAT/enhancer-binding protein α (C/EBPα) expression and preadipocyte differentiation.

doi: 10.1210/en.2014-1565

Figure Lengend Snippet: Figure 1. siRNA knockdown of HDAC1 in NIH 3T3 cells increases the efficiency of preadipocyte differentiation in a Dex-dependent manner. A, Western blot analysis of HDAC1, HDAC2, and ACTIN, 48 hours after transfection of C/EBP-expressing NIH 3T3 cells with either no siRNA (), scrambled (Scr), or HDAC1 siRNA. B, Immunoblot quantification of HDAC1 protein expression levels from A (values are normalized to ACTIN expression, n 3, SD). A Student’s t test was performed in all data, where *, P .05. C, Western blot analysis of the adipocyte marker ADIPSIN in siRNA-treated NIH 3T3 cells on day 4 of differentiation. Two days after NIH 3T3 cells were transfected with the siRNA, they were stimulated to differentiate with either MI (MIX and insulin) or MID (MI and Dex) for 48 hours, followed by 2 days of incubation in media with just insulin. Three independent experiments were performed. D, Quantification of C. ADIPSIN expression levels were quantified relative to the results of the NIH 3T3 cells treated with Scr siRNA and MID (n 3, SD). E, Qualitative assessment of adipogenesis by Oil red O staining of the NIH 3T3 cells with either Scr or HDAC1 siRNA 4 days after treatment with MID. Three independent experiments were performed. F, qPCR was performed for C/ebp and Ppar. RNA was harvested from the NIH 3T3 cells transfected with either Scr or HDAC1 siRNA, 48 hours after initiation of differentiation with MI or MID. Only the qPCR values for the MID samples are shown, because the values for the MI samples were too low to be accurately quantified. qPCR values are presented relative to the level in the Scr MID sample and were normalized to -Actin values (n 3 duplicates, SEM). A Student’s t test was performed in all data, where *, P .05.

Article Snippet: Western blot analysis, antibodies, and Oil red O staining NIH 3T3 cells were harvested on day 4 of differentiation, 3T3-L1 cells were harvested on day 7, and differentiation efficiency was examined via Western blot analysis of known adipogenic marker using ADIPSIN P-16 antibody from Santa Cruz Biotechnology, Inc. All Western blottings were quantified using densitometry through the ImageQuant program, and the values were normalized to ACTIN protein expression levels (ACTIN H-300 antibody; Santa Cruz Biotechnology, Inc).

Techniques: Knockdown, Western Blot, Transfection, Expressing, Marker, Incubation, Staining

Journal: Endocrinology

Article Title: Inactivation of histone deacetylase 1 (HDAC1) but not HDAC2 is required for the glucocorticoid-dependent CCAAT/enhancer-binding protein α (C/EBPα) expression and preadipocyte differentiation.

doi: 10.1210/en.2014-1565

Figure Lengend Snippet: Figure 3. siRNA knockdown of HDAC2 in NIH 3T3 cells does not alter preadipocyte differentiation. A, Western blot analysis of HDAC2, HDAC1, and ACTIN, 48 hours after transfection of C/EBP-expressing NIH 3T3 cells with either scrambled (Scr) or HDAC2 siRNA. The HDAC2 blot is quantified in the lower panel similarly to Figure 1B. B, Western blot analysis of the adipocyte marker ADIPSIN in siRNA-treated NIH 3T3 cells on day 4 of differentiation, as in Figure 1C. Three independent experiments were performed. C, Oil red O staining of the 3T3-L1 cells with either Scr or HDAC2 siRNA 4 days after treatment with MID. Three independent experiments were performed.

Article Snippet: Western blot analysis, antibodies, and Oil red O staining NIH 3T3 cells were harvested on day 4 of differentiation, 3T3-L1 cells were harvested on day 7, and differentiation efficiency was examined via Western blot analysis of known adipogenic marker using ADIPSIN P-16 antibody from Santa Cruz Biotechnology, Inc. All Western blottings were quantified using densitometry through the ImageQuant program, and the values were normalized to ACTIN protein expression levels (ACTIN H-300 antibody; Santa Cruz Biotechnology, Inc).

Techniques: Knockdown, Western Blot, Transfection, Expressing, Marker, Staining

Journal: Endocrinology

Article Title: Inactivation of histone deacetylase 1 (HDAC1) but not HDAC2 is required for the glucocorticoid-dependent CCAAT/enhancer-binding protein α (C/EBPα) expression and preadipocyte differentiation.

doi: 10.1210/en.2014-1565

Figure Lengend Snippet: Figure 2. siRNA knockdown of HDAC1 in the 3T3-L1 preadipocyte cell line increases differentiation in a Dex-dependent manner. A, Western blot analysis of HDAC1and ACTIN (upper panel), 48 hours after transfection of 3T3-L1 cells with either scrambled (Scr) or HDAC1 siRNA. The quantification of the blot is shown in the lower panel and quantified as in Figure 1B. B, Western blot analysis of the adipocyte marker ADIPSIN in siRNA-treated 3T3-L1 cells on day 7 of differentiation (upper panel). Two days after 3T3-L1 cells were transfected with the siRNA, they were stimulated to differentiate with either MI (MIX and insulin) or MID (MI and Dex) for 48 hours, followed by 5 days of incubation in media with just insulin. Three independent experiments were performed. Quantification of the blot is shown in the lower panel and quantified as in Figure 1D. C, Qualitative assessment of adipogenesis by Oil red O staining of the 3T3-L1 cells with either Scr or HDAC1 siRNA 4 days after treatment with MID. Three independent experiments were performed.

Article Snippet: Western blot analysis, antibodies, and Oil red O staining NIH 3T3 cells were harvested on day 4 of differentiation, 3T3-L1 cells were harvested on day 7, and differentiation efficiency was examined via Western blot analysis of known adipogenic marker using ADIPSIN P-16 antibody from Santa Cruz Biotechnology, Inc. All Western blottings were quantified using densitometry through the ImageQuant program, and the values were normalized to ACTIN protein expression levels (ACTIN H-300 antibody; Santa Cruz Biotechnology, Inc).

Techniques: Knockdown, Western Blot, Transfection, Marker, Incubation, Staining

Journal: Endocrinology

Article Title: Inactivation of histone deacetylase 1 (HDAC1) but not HDAC2 is required for the glucocorticoid-dependent CCAAT/enhancer-binding protein α (C/EBPα) expression and preadipocyte differentiation.

doi: 10.1210/en.2014-1565

Figure Lengend Snippet: Figure 5. HDAC1D181A potentiates 3T3-L1 cells ability to differentiate into mature adipocytes in a Dex-dependent manner. A, Western blot analysis of the adipocyte marker ADIPSIN in 3T3-L1 cells stably expressing empty plasmid (pLXSN), HA-Flag-HDAC1WT, or HA-Flag- HDAC1D181A on day 7 of differentiation. The cells were stimulated to differentiate with either MI (MIX and insulin) or MID (MI and Dex) for 48 hours, followed by 5 days of incubation in media with just insulin. Immunoblot of HA is shown to ensure similar expression of both HDAC1 constructs. Three independent experiments were performed. B, Oil red O staining of the 3T3-L1 cells stably expressing empty plasmid (pLXSN), HA-Flag-HDAC1WT, or HA-Flag-HDAC1D181A 4 days after treatment with MID. Three independent experiments were performed. C, qPCR was performed for C/ebp and Ppar mRNA from the 3T3-L1 cells stably expressing either HA-Flag-HDAC1WT or HA-Flag- HDAC1D181A 48 hours after initiation of differentiation with MI or MID. Only the MID values are shown, because the values for the MI samples were too low to be accurately quantified. The qPCR values are presented relative to the level in the empty plasmid (pLXSN condition) MID sample and were normalized to -Actin values (n 3 duplicates, SEM). A Student’s t test was performed in all data where, where *, P .05.

Article Snippet: Western blot analysis, antibodies, and Oil red O staining NIH 3T3 cells were harvested on day 4 of differentiation, 3T3-L1 cells were harvested on day 7, and differentiation efficiency was examined via Western blot analysis of known adipogenic marker using ADIPSIN P-16 antibody from Santa Cruz Biotechnology, Inc. All Western blottings were quantified using densitometry through the ImageQuant program, and the values were normalized to ACTIN protein expression levels (ACTIN H-300 antibody; Santa Cruz Biotechnology, Inc).

Techniques: Western Blot, Marker, Stable Transfection, Expressing, Plasmid Preparation, Incubation, Construct, Staining

Journal: Adipocyte

Article Title: Bisphenol A increases aP2 expression in 3T3L1 by enhancing the transcriptional activity of nuclear receptors at the promoter

doi: 10.4161/adip.28436

Figure Lengend Snippet: Figure 1. Bisphenol A (BPA) treatment of 3T3-L1 cells induces adipocyte differentiation markers and lipid accumulation. ( A ) Oil red O lipid staining of 3T3-L1 cells at day 8 treated with the indicated concentrations of BPA. ( B ) Western blot analyses of aP2 and adipsin at day 8. ( C ) Fold over MI ethanol control of the western blots normalized to β-actin levels and quantified with BioRad ImageLab software ( n = 3). ( D ) aP2 mRNA levels over control normalized to β-actin mRNA after 24 h treatment ( n = 5). Error bars indicate SD (* P < 0.05, ** P < 0.01).

Article Snippet: Equal amounts of protein (10–30 μg) were resolved by SDS-PAGE and transferred to polyvinylidene membrane (Bio-Rad) for western analysis with the primary antibodies for aP2 (adipocyte fatty acid binding protein 4 [FABP4]) (R&D), adipsin (P-16) (Santa Cruz), and β-actin (13E5) (Cell Signaling).

Techniques: Staining, Western Blot, Control, Software

Journal: Adipocyte

Article Title: Bisphenol A increases aP2 expression in 3T3L1 by enhancing the transcriptional activity of nuclear receptors at the promoter

doi: 10.4161/adip.28436

Figure Lengend Snippet: Figure 6. High dose BPA upregulates aP2 and adipsin in the absence of PPARγ or C/EBPα induction. ( A ) mRNA levels of aP2 (left panel) and adipsin (right panel) standardized against control β-actin mRNA levels and expressed as fold over MI + vehicle control at the indicated time points ( n = 5). ( B ) Protein levels of the indicated proteins by western blot. ( C ) Analysis of western blots of aP2 and adipsin protein levels quantified using the BioRad ImageLab software represented as fold increase of aP2 or adipsin respectively, over MI + vehicle and normalized to β-actin protein levels ( n = 3). Error bars represent SD (* P < 0.05, ** P < 0.01).

Article Snippet: Equal amounts of protein (10–30 μg) were resolved by SDS-PAGE and transferred to polyvinylidene membrane (Bio-Rad) for western analysis with the primary antibodies for aP2 (adipocyte fatty acid binding protein 4 [FABP4]) (R&D), adipsin (P-16) (Santa Cruz), and β-actin (13E5) (Cell Signaling).

Techniques: Control, Western Blot, Software