Cells from a 10-cm dish were resuspended in PBS. The cytoplasmic and nuclear fraction were collected using nuclear and cytoplasmic protein extraction kit (P0028, Beyotime, Hangzhou, China). For analysis of ZEB1 DNA binding, EMSA was used and performed as previously described [49 ]. Double-stranded oligonucleotide probes were synthesized (Beyotime, Hangzhou, China) to contain the E-box site sequences from the promoters of the indicated target genes (ATM). The oligos were end labeled with biotin using biotin 3′end DNA labeling kit (GS008, Beyotime, Hangzhou, China). EMSA was performed by incubating 5 μg of nuclear extract with 4 nmol biotin-labeled DNA probe. Absence of nuclear extract, competition with 100-fold molar excess unlabeled DNA prob or non-specific oligo served as controls. Supershift studies were performed by pre-incubation with the specified antibodies for 30 min. All EMSA assays were performed by chemiluminescent EMSA kit (GS009, Beyotime, Hangzhou, China). Complexes were separated by electrophoresis on non-denaturing 6.5% acrylamide gel. Sequences of EMSA probes are provided as follow: E-box: forward 5′-TGGCATTTCACACCTCTACACTGGACG′, reverse 5′-CGTCCAGTGTAGAGGTGTGAAATGCCA-3′; Non-specific: forward 5′-TCGAGTTGATGTAACCGACTCAGGCACT′, reverse 5′-AGTGCCTGAGTCGGTTACATCAACTCGA-3′.
P0028
Manufactured by Beyotime
Sourced in China
The P0028 is a laboratory equipment designed for general use in scientific and research settings. It serves as a reliable tool for various experimental and analytical tasks. The core function of the P0028 is to provide a controlled environment for conducting experiments or analyses, ensuring consistent and accurate results.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions)
Biotin 3 end dna labeling kit, by Beyotime (1 mentions)
P0011, by Beyotime (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab215715, by Abcam (1 mentions)
Ab228653, by Abcam (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Sc 460, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Anti sirt6, by Cell Signaling Technology (1 mentions)
C3601, by Beyotime (1 mentions)
P0033, by Beyotime (1 mentions)
Ripa lysis buffer, by Keygen Biotech (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Wl02400, by Wanlei (1 mentions)
Wl02635, by Wanlei (1 mentions)
30 protocols using p0028
1
Assessing ZEB1 DNA Binding via EMSA
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from tissues and cells with radio-immunoprecipitation assay lysis buffer (P0013B, Beyotime) containing 100 mM phenylmethylsulphonyl fluoride. The nuclear and cytoplasmic protein was extracted with kit (P0028, Beyotime) according to the instructions, followed by measurement of protein concentration with a bicinchoninic acid kit (P0011, Beyotime). After separation by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred onto a polyvinylidene fluoride membrane (1620177, Bio-Rad). Next, the membrane was blocked with 5% skimmed milk powder for 1 h and underwent overnight incubation at 4 °C with primary rabbit antibodies against β-actin (4970, 1:5000, Cell Signaling Technology, Beverly, MA), TGF-β (ab215715, 1:1000, Abcam, Cambridge, UK) and SREBP2 (ab228653, 1:1000, Abcam). The following day, the membrane was incubated with horseradish peroxidase-labeled secondary antibody goat anti-rabbit IgG (ab6721, 1:5000, Abcam) at room temperature for 1 h. Afterwards, the immunocomplexes on the membrane were visualized using enhanced chemiluminescence reagent (1705062, Bio-Rad). Image exposure was performed on Image Quant LAS 4000 C gel imager (GE company, Fairfield, CT). With β-actin serving as the internal reference, protein expression was quantified utilizing the Image J software.
3
Protein Extraction Using Commercial Kit
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The nuclear and cytoplasmic protein extraction was conducted by following the manufacturer’s instructions of the commercial kit (P0028, Beyotime).
4
Smad2 Acetylation by Sirt6 in TGF-β Signaling
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HEK293 cells were transfected with Smad2 with or without Sirt6 and then 48 hours later were treated with TGF‐β1, and the nuclear fraction was prepared by using a kit (Beyotime, P0028). The nuclear protein was immunoprecipitated with antiacetylation antibody and Protein A/G PLUS‐agarose (sc‐2003, Santa Cruz Biotechnology) overnight at 4°C. Immunoprecipitated protein was resolved by 10% SDS‐PAGE gels for Coomassie blue staining. The gels were used for mass spectrometry (MS).
5
Western Blot Analysis of Cellular Signaling
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Proteins were extracted from the cells at the indicated times using SDS lysis buffer (P0013G, Beyotime, China). Nuclear and Cytoplasmic Extraction Reagents (P0028, Beyotime, China) were used to separate the cytoplasmic and nuclear extracts from cultured cells. The protein samples (30 μg) were subjected to 4–20% polyacrylamide gels (JC-PE022/JC-PE022R, Genshare biological, China) to be electrophoresed and transferred to 0.22 μm PVDF membranes (ISEQ00010, Merck Millipore, USA) at 90 V for 1.5 h. Membranes were blocked with 5% skim milk for about 1 h at room temperature and then incubated with primary antibodies against HPV 16 E6 (sc-460, Santa Cruz, USA; 1:200), IκBα (4814, CST, USA; 1:1000), p-IκBα (Ser 32/36) (9246, CST, USA; 1:1000), p65 (10745-1-AP, Proteintech, USA; 1:1000), p-p65 (Ser536) (3031, CST, USA; 1:1000), p-p65 (Ser468) (3039, CST, USA; 1:1000), H3 histone (4499, CST, USA; 1:1000), Akt (10176-2-AP, Proteintech, USA; 1:1000), p-Akt (Ser473) (4060, CST, USA; 1:1000), p-Akt (Thr308) (13038, CST, USA; 1:1000), GAPDH (Proteintech, USA; 1:10,000), and β-actin (3700, CST, USA; 1:1000) at 4 °C overnight. Thereafter, the membranes were probed with IR Dye-labeled secondary antibodies (800CW, LI-COR Bioscience, USA; 1:10,000) and the signals were observed using an Odyssey Infrared Imaging System (LI-COR Bioscience, USA).
6
Cytoplasmic and Nuclear Protein Extraction
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Cytoplasmic and nuclear protein extraction was performed using a commercial kit (P0028, Beyotime) following the manufacturer's instructions. Cells were harvested and suspended by equal volume solution A containing 1% PMSF, and cell lysates were mixed with 10 μl solution B by vortex and placed on ice for 1 min. Tissue sample fragments were homogenized with a mixture of solutions A and B by a glass homogenizer. Supernatant (cytoplasmic protein) was obtained after 12,000 rpm centrifugation for 5 min; 50 μl solution C was used to suspend the sediment. Samples were mixed by vortex for 30 s every 2 min for 15 times, and supernatant (nuclear protein) was collected after centrifugation.
7
Isolation and Immunoprecipitation of Nuclear Proteins
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Using a cytoplasmic and nuclear protein isolation kit (Beyotime, P0028), nuclear extracts were obtained from chondrocytes. Chondrocytes were then lysed with non-denaturing NP-40 lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma). An amount of 500 μg protein was immunoprecipitated with anti-Sirt6 (Cell Signaling Technology) or anti-total-STAT5 antibody (Cell Signaling Technology) and Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology) overnight at 4 °C. Beads with the immune-precipitated complex were washed with lysis buffer three times before denaturing in lysis buffer at 97 °C for 10 min. Nuclear extracts and immunoprecipitated protein were resolved by SDS-PAGE. Western blot was then performed.
8
Nuclear Protein Binding Assay
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Nuclear protein extracts were prepared following manufacturer's instructions (P0028; Beyotime Biotechnology). Nuclear proteins (2 μg) were incubated for 20 min at room temperature with a biotin-labeled AP-1 consensus oligonucleotide (Sigma) (5›-CGC TTG ATG ACT CAG CCG GAA-3›, 3›-GCG AAC TAC TGA GTC GGC CTT-5›) as per the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). Reaction mixtures were electrophoresed in a 6% polyacrylamide gel, transferred, DNA cross-linked to a nylon membrane (Amersham; GE Healthcare, UK) and then probed with an HRP–conjugated streptavidin antibody (1:300). The membranes were washed with washing buffer for 5 min 3 times and incubated in equilibration buffer for 5 min before ECL detection.
9
Nuclear Protein Extraction from Kidney
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Nuclear fractions were extracted from fresh kidney tissues by using a nuclear protein extraction kit (P0028, Beyotime, CN) according to the manufacturer's instructions.
10
Nuclear Protein Extraction Protocol
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Nuclear protein was extracted using a commercial nuclear protein and cytoplasmic protein extraction kit P0028 (Beyotime, Haimen, China) in accordance with the manufacturer’s instructions. Briefly, 1 mM tissue homogenate (60 mg/200 μL) was transferred into a centrifuge tube and ice bath for 15 min at 4 °C. The homogenate was then centrifuged at 1500× g for 5 min at 4 °C. PMSF protein extraction reagent A (60 mg/200 μL) was added to the collected precipitate, and the sample was vortexed for 5 s. The sample was added to 10 μL protein extraction reagent B and was centrifuged (12,000–16,000× g, 5 min, 4 °C). The precipitate was then added to 50 μL nuclear protein extraction reagent and vortexed for 15–30 s per 1–2 min for 30 min. Finally, samples were centrifuged at 12,000–16,000× g for 10 min at 4 °C. The harvested precipitate contained nuclear protein. Protein concentrations were calculated using a BCA Protein Assay Kit (Pierce 23225; Rockford, IL, USA).
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