Nuclear extraction kit

For detecting of NFkB p65 levels, an ELISA based assay (NFkB p65 Transcription Factor Assay Kit -ab133112, ABCAM, Cambridge, UK) was used, according to the manufacturer’s instructions. For extract preparation, a Nuclear Extraction Kit, ab113474, ABCAM, Cambridge, UK) was used, according to the manufacturer’s instructions.

The nuclear protein fraction was extracted from spermatozoa using the Nuclear Extraction Kit following the manufacturer’s recommendations (Abcam, United States). In brief, 2 × 10⁶ testicular, caput, corpus, and cauda spermatozoa were suspended in 100 µl of 1× Pre-Extraction Buffer supplied with the kit. After a 10-min incubation on ice, the cell suspension was vortexed vigorously for 10 s and centrifuged at 10,000 g for 1 min. The cytoplasmic fraction was carefully removed from the nuclear pellet and stored at −20°C for later use. Twenty μl of Extraction Buffer containing DTT and Protease Inhibitor Cocktail were added to the nuclear pellet and incubated on ice for 15 min with a 5-s vortex step every 3 min. The suspension was collected after centrifugation at 14,000 g for 15 min at 4°C. Protein concentration was determined using the Bradford reagent (Biorad, CANADA) following the standard protocol.

AMPK activity was performed using an AMPK kinase assay kit (CycLex, Nagano, Japan) and quantified by measuring absorbance at 450 nm using a microplate reader [19 (link)]. SIRT1 activity was measured using a nuclear extraction kit (Abcam, Cambridge, UK), and then the nuclear fraction was extracted from cells using the SIRT1 activity assay kit (Abcam, Cambridge, UK). Fluorescence intensity was detected at 340 nm excitation and 460 nm emission, respectively, using a microplate fluorescence reader.

Nuclear extraction was conducted using the Abcam Nuclear Extraction Kit according to the manufacturer’s instructions. For experiments on cultured cells, confluent cells were scraped and incubated on ice for 10 min in pre-extraction buffer containing dithiothreitol (DTT) and protease inhibitor cocktail (PIC), then centrifuged at 12,000 rpm for 1 min. The cytoplasmic supernatant was removed, and the pellets incubated on ice for 15 min in nuclear extraction buffer containing DTT and PIC, then sonicated 3 times for 10 s. The extract was then centrifuged at 14,000 rpm for 10 min at 4 °C and the supernatant removed for protein quantification using the Pierce Coomassie Protein Assay Kit (ThermoFisher). For experiments on murine liver, samples were placed in a glass homogenizer containing pre-extraction buffer with DTT and PIC and homogenized manually. Homogenized samples were transferred to microcentrifuge tubes and incubated on ice for 15 min then centrifuged at 10,000 rpm for 10 min at 4 °C. The cytoplasmic supernatant was removed, and nuclear extraction continued as for cultured cells.

Nuclear lysates of 10⁷ cells were prepared using the Nuclear Extraction Kit (ab113474, Abcam, Cambridge, UK). Upon formaldehyde crosslinking and sonication-mediated DNA shearing, H3K9ac, BRD4, Foxp1, and IgG immunoprecipitates in nuclear extracts were prepared using EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kits (Millipore, Temecula, CA, USA) along with specific antibodies, according to the manufacturer’s instructions. In brief, 1 μg of antibodies, IgG, and anti-RNA polymerase, together with protein G magnetic beads, were added to specimens and incubated at 4 °C with rotation for 16 h. Protein G-chromatin complexes were harvested using a magnetic separator and washed using Low Salt, High Salt, and LiCl Immune Wash Buffers. Specimens were mixed with Proteinase K in ChIP Elution Buffer and incubated at 62 °C for 2 h and 95 °C for 10 min. DNA was harvested and concentrated using spin columns. A total of 1 ng DNA was mixed with PCR mixtures and Cy3-conjugated primers for Runx2 (−942~+28 bp; ENSMUSG00000039153), PPARγ2 (−1996~−1731 bp; ENSMUSG00000000440), Foxp1 (−249~+32 bp; ENSMUSG00000030067) promoters and positive control GADPH promoter (ENSMUSG00000207654). The enrichment of H3K9ac, BRD4, Foxp1, and IgG in Runx2 and PPARγ2 promoter was expressed as % input DNA.

Nuclear protein was extracted using the nuclear extraction kit (Abcam) as follows: small pieces of tissue were weighed and cut. Tissue was homogenized with pre-extraction buffer containing dithiothreitol (DTT) solution, and centrifuged, followed by removal of supernatant. The extraction buffer (containing DTT solution and a protease inhibitor cocktail) was added to the nuclear sediment, and the extract was incubated on ice while being vortexed. Finally, the suspension was centrifuged and supernatant was collected.

Cells were lysed using RIPA buffer (Heart, Xian, China). Cell lysates containing 30 μg of total protein were then subjected to SDS–PAGE (Beyotime, Shanghai, China) and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C (anti-Zic2, Axin2, APC, active-β-Catenin(ser45), β-Catenin, Flag, and c-Myc, 1:1000 dilution; Cyclin D1 and GAPDH, 1:5000). The membrane was then washed six times with TBST buffer for 5 min each and incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) was added to visualize the protein bands. The antibodies against GAPDH were purchased from Santa Cruz (Dallas, TX, USA), the antibodies against Zic2, Cyclin D1, CD44, Axin2, and APC were purchased from Abcam (Cambridge, MA). The antibodies against active-β-Catenin(ser45), β-Catenin, and GSK-3β were purchased from Cell Signaling Technology (Danvers, MA, USA), and the antibodies against Flag was purchased from Proteintech (Rosemont, USA). Nuclear extract was prepared with the protocol in the Nuclear Extraction Kit (Abcam, Cambridge, MA, USA).