For protein immunoprecipitation, cells (1 × 107) overexpressed Flag-tagged NUPR1 were harvested, and lysate samples containing 2000 μg total protein were immunoprecipitated with 4 μl M2-Flag (Sigma) or SREBP1 (Santa Cruz Biotechnology) respectively at 4 °C overnight, followed by incubation with 40 μl Protein A/G plus agarose beads (Santa Cruz Biotechnology) at 4 °C for 4 h. Beads were washed and boiled in 40 µl of loading buffer, and then western blotting was conducted using SREBP1 (Proteintech, 14088-1-AP) or anti-NUPR1 (Proteintech, 15056-1-AP). The protein of input used in Fig. 4-A was about 280 µg and 8 μl used for IP.
Srebp1
Manufactured by Proteintech
Sourced in China
SREBP1 is a transcription factor that regulates the expression of genes involved in lipid and cholesterol biosynthesis. It plays a central role in the homeostatic control of cellular lipid levels.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Proteintech (3 mentions)
Gapdh, by Proteintech (2 mentions)
P erk1 2, by Proteintech (2 mentions)
Srebp1, by Santa Cruz Biotechnology (1 mentions)
Flag m2, by Merck Group (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Insulin receptor β, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
P ampkα, by Cell Signaling Technology (1 mentions)
Cytochrome c, by ABclonal (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Goat anti mouse igg, by Wuhan Servicebio Technology (1 mentions)
Goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Fabp4, by Proteintech (1 mentions)
Gapdh, by Wuhan Servicebio Technology (1 mentions)
Pparγ, by Proteintech (1 mentions)
Anti ampkα1, by Cell Signaling Technology (1 mentions)
Ampk inhibitor compound c, by Selleck Chemicals (1 mentions)
10 protocols using srebp1
1
Immunoprecipitation of Flag-tagged NUPR1
2
Western Blot Analysis of Protein Expression
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RIPA lysis buffer (Beyotime) was adopted to acquire protein sample. Then the equal amount protein samples were loaded on 8–12% polyacrylamide gel for electrophoresis separation, followed by blotting onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, USA). Blocking was performed by incubation in 5% bovine serum albumin (BSA, Biosharp, Guangzhou, China) for 1 h. Then the membranes were probed with primary antibodies SREBP1 (1:1000, Proteintech, China), CYP17 (1:500, Proteintech), CYP19 (1:500, Proteintech), AMPKα (1:1000, Cell Signaling Technology, USA), p-AMPKα (1:1000, Cell Signaling Technology), p-AKT (1:2000, Cell Signaling Technology), AKT (1:1000, Cell Signaling Technology), p-IR (1:1000, Cell Signaling Technology), insulin receptor β (IR, 1:1000, Cell Signaling Technology), cytochrome C (1:1000, Abclonal, China), β-actin (1:2000, Proteintech) at 4 ℃ overnight. Goat anti-rabbit or anti-mouse IgG (1:10000, Proteintech) was used as secondary antibody. The antigen–antibody complexes were visualized with ECL Plus reagent (7 sea biotech, Shanghai, China).
3
Protein Expression Profiling in PSCs and Preadipocytes
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In PSCs, the expression levels of MYOG, MYOD, MHC and CXXC5 protein were detected. The protein expression levels of FABP4, PPARγ, SREBP1 and FASN were detected in preadipocytes. Transfected cells were lysed in RIPA buffer with 1% PMSF. 5×Buffer was added to the sample and denatured at 100 °C for 10 min. SDS-PAGE gel electrophoresis was performed at 80 V 30 min, 120 V 90 min. Then transferred them onto a PVDF membrane and non-specific binding was blocked with 5% non-fat milk in PBS for 1 h. Then, they were incubated with 1:1000 diluted polyclonal rabbit MYOG (Abclonal, China), MYOD (Proteintech, China), CXXC5 (Bioss, China), 1:500 diluted polyclonal mouse MHC (DSHB, America), 1:1000 diluted polyclonal rabbit FABP4 (Proteintech, China), PPARγ (Proteintech, China), SREBP1 (Proteintech, China) and FASN (Proteintech, China) at 4 ℃ overnight. The blots were subsequently incubated with secondary antibody (1:10000) for 1 h. Secondary antibody include goat anti-mouse IgG (Servicebio, China) and goat anti-rabbit IgG (Servicebio, China). GAPDH (Servicebio, China) was used as an endogenous protein for normalization. Image J software was used to conduct quantitative analysis of western blot results according to the gray value of the strip.
4
Investigating AMPK Regulation by Apigenin
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Palmitic acid and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA. The apigenin standard was purchased from Beijing Solarbio Technology Co. The AMPK inhibitor compound C was purchased from Selleck Chemicals LLC. Anti-phospho-AMPK (P-AMPK; Thr172; cat. no. 2535) was purchased from Cell Signaling Technology, Inc., anti-AMPKα1 (cat. no. 10929-2-AP), SREBP-2 (cat. no. 14508-1-AP), FAS (cat. no. 13098-1-AP), HMGCR (cat. no. 13533-1-AP) and β-actin (cat. no. 66009-1-lg) were purchased from Proteintech Group Inc., SREBP-1 (cat. no. ab3259) was purchased from Abcam. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L; cat. no. SA00001-1) and HRP-conjugated goat anti-rabbit IgG (H + L; cat. no. SA00001-2) secondary antibodies were obtained from Proteintech Group Inc.
5
Protein Expression Analysis Workflow
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Cells were harvested and lysed in RIPA buffer containing protease inhibitor (Solarbio). Total protein concentration was measured by the BCA assay kit (Solarbio). The protein labels were visualized using the ECL detection system (Thermo). The primary antibodies of SIRT1 (1:500), SREBP1(1:500), ACC (1:500), FASN (1:500), and GAPDH (1:1,000) were purchased from Proteintech.
6
Protein Extraction and Western Blot Analysis
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The proteins were extracted using EBC lysis buffer (50 mM, Tris pH 8.0, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Selleck). The protein concentrations were measured using a Bradford Protein Assay Kit (Bio-Rad). Total proteins (20 μg) were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Bio-Rad). After blocking with 5% non-fat milk in tris-buffered saline with tween at room temperature for 30 min, the membranes were probed with antibodies. The antibody against FBW7 was purchased from Bethyl, and p-FBW7 was custom-ordered from Abclonal Technology. The PGC1α, ERK1, HA and tubulin antibodies were purchased from Santa Cruz, and p-ERK, Mcl-1 and the phospho-TP monoclonal antibodies were purchased from Cell Signaling Technology, while c-Myc, cyclin E, Notch1, c-Jun, p-AKT and p-RSK antibodies were all purchased from Abcam. The GST, Myc, SREBP1 and KLF-5 antibodies were purchased from Proteintech. The Flag antibody was purchased from Sigma-Aldrich. All secondary antibodies were purchased from Dako. After incubation with primary and secondary antibodies, immunoblots were incubated with an enhanced chemiluminescence detection kit (Millipore) and visualized in a dark room.
7
Protein Expression Analysis in Tissue Lysates
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Tissue and cell lysates were prepared using RIPA buffer (20 mM Tris-Cl pH 7.5, 140 mM NaCl, 1 mM CaCl2 and MgCl2, 10 mM NaF, 1% NP-40, 10% glycerol, 2 mM Na-Vanadate, and 1 mM PMSF). Proteins were subjected to SDS-PAGE and were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Immunoblotting was performed with the following primary antibodies, according to the manufacturer’s protocol: KLF9 (Invitrogen; Cat: 701888; Clone name: 5H16L7; Dilution: 1:1000), KLF9 (Abcam; Cat: ab227920; Dilution: 1:1000), PGC1α (Millipore; Cat: AB3242; Dilution: 1:1000), UCP1 (Abcam; Cat: ab10983; Dilution: 1:1000), SERCA2(ABclonal; Cat: A11692; Clone name: ARC0679; Dilution: 1:1000), β-Actin (ABclonal; Cat: AC026; Clone name: ARC5115-01; Dilution: 1:20,000), ARG1 (ABclonal; Cat: A4923; Clone name: ARC1164; Dilution: 1:1000), SREBP1 (ABclonal; Cat: A15586; Dilution: 1:1000), HDAC1 (ABclonal; Cat: A0238; Dilution: 1:1000), HDAC2 (ABclonal; Cat: A2084; Dilution: 1:1000), SIN3A (Proteintech; Cat: 14638-1-AP; Dilution: 1:1000), STAT3 (Santa Cruz Biotechnology; Cat: sc-8019; Dilution: 1:50), P-STAT3 (Santa Cruz Biotechnology; Cat: sc-8059; Dilution: 1:50), p65 (Cell Signaling Technology; Cat: #8242; Clone name: D14E12; Dilution: 1:1000), P-p65 (Cell Signaling Technology; Cat: #3033; Clone name: 93H1; Dilution: 1:1000).
8
Protein Extraction and Western Blot Analysis
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High-efficiency RIPA tissue/cell lysate (Beijing Soleibo Technology Co., Ltd., China) (containing PMSF) was used to lyse liver cancer tissue or cells to extract proteins, and then they were separated on a 10% or 12% gel with SDS-polyacrylamide. Samples (20 μg) were wet-transferred into 0.22 μm PVDF membranes (Millipore, Burlington, MA, USA) and covered with 5% nonfat milk for 2 hours at room temperature, followed by incubation with primary antibodies overnight (SREBP1 1:1000, Cat No. 14088-1-AP, Proteintech, Wuhan, China), (ACC1 1:1000, Cat No. 21923-1-AP, Proteintech, Wuhan, China), (FASN 1:1000, Cat No. 10624-2-AP, Proteintech, Wuhan, China), (ACLY 1:2000, Cat No. 15421-1-AP, Proteintech, Wuhan, China), (P38 1:1000, Cat No. 14064-1-AP, Proteintech, Wuhan, China), (P-P38 1:1000, Cat No. 28796-1-AP, Proteintech, Wuhan, China), (ERK1/2 1:1000, Cat No. 11257-1-AP, Proteintech, Wuhan, China), (P-ERK1/2 1:1000, Cat No. 28733-1-AP, Proteintech, Wuhan, China), (GAPDH 1:10000, Catalog No. 10494). The membranes were washed 3 times in TBS with 0.1% Tween-20 buffer for 10 min each, reacted with secondary antibody for 2 h at room temperature, and then washed 3 times in TBST buffer for 10 min each minute. Bands were visualized with enhanced chemiluminescence Western blot detection reagents (Advansta, Inc., Menlo Park, CA, USA), and relative protein levels were quantified by using ImageJ.
9
Immunofluorescent Quantification of Signaling Proteins
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Immunofluorescence staining and microscopy were performed as described previously [23 (link)], and the concentrations of antibodies were as follows: SREBP1 1:100 (Cat No. 14088-1-AP, Proteintech, Wuhan, China), P38 1:250 (Cat No. 14064-1-AP, Proteintech, Wuhan, China), P-P38 1:250 (Cat No. 28796-1-AP, Proteintech, Wuhan, China), ERK1/2 1:250 (Cat No. 11257-1-AP, Proteintech, Wuhan, China), and P-ERK1/2 1:100 (Cat No. 28733-1-AP, Proteintech, Wuhan, China). To perform semiquantitative analysis, the fluorescence intensity of immunofluorescence was measured via ImageJ.
10
Protein expression analysis by Western blot
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The cell samples were lysed by cell lysate (Beyotime, P0013) and boiled for 10 min to obtain the protein samples, which were then electrophoresed and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, ISEQ00010). The samples were blocked in 5% non-fat milk for 1 hour at room temperature and then treated with the primary antibodies of HCAR1 (Abcam, ab124010. 1:300), MCT1 (PROTEINTECH, 20139-1-AP. 1:5000), AMPK (Cell Signaling Technology, #5832. 1:1000), p-AMPK (Cell Signaling Technology, #2535. 1:1000), SREBP1 (PROTEINTECH, 14088-1-AP. 1:800), SCD1 (Cell Signaling Technology, #2794. 1:1000), ACSL4 (abcam, #ab155282. 1:5000), GPX4 (abcam, ab125066. 1:1000), FSP1(PROTEINTECH, 20886-1-AP. 1:1000) and b-actin(bioss, bs-0061R. 1:5000), following the standard procedures from manufacturer's instructions. Afterward the samples were incubated with secondary antibadies (Beyotime, A0208) and washed three times. Electrochemiluminescence (Beyotime, P0018FS) was captured on a gel imaging system (Biorad ChemiDoc MP).
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