Ab227383

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before (Li et al., 2019b , f , 2020a (link), b , c (link); Shao et al., 2019 (link); Weng et al., 2019 (link); Chen et al., 2020a ; Wu X. et al., 2020 (link); Hong et al., 2021 (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Santa Cruz, sc-10768), anti-Sp1 (Abcam, ab227383), or pre-immune IgG. Precipitated genomic DNA was amplified by real-time PCR with the following primers: SCAP proximal promoter, 5′-ATACTTCCCTCCGGTGTCCAC-3′ and 5′-ACCTCTCACCTCCACCTTTAC-3′; SCAP distal promoter, 5′-AAATGCGAGGACATGTACAATAC-3′ and 5′-ATTTAAAAGCTAAGTTGAC-3′. A total of 10% of the starting material is also included as the input. Data are then normalized to the input and expressed as % recovery relative the input as previously described (Chen et al., 2020b (link), c (link)). All experiments were performed in triplicate wells and repeated three times.

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before (Chen et al., 2020a (link), b (link), c (link); Dong et al., 2020 (link); Fan et al., 2020 (link); Li et al., 2020a (link), b (link), c (link); Lv et al., 2020 (link); Mao et al., 2020 (link); Sun et al., 2020 (link); Wu et al., 2020 (link); Yang et al., 2020 (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mMNaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Santa Cruz, sc-10768), anti-NF-κB (Santa Cruz, sc-372), anti-Sp1 (Abcam, ab227383), or pre-immune IgG.

Total protein was extracted by suspending the cell pellet in a cell lysis buffer containing 50 mM Tris‐HCl (pH 7.4), 150 mM NaCl, 1% Triton X‐100, 0.1% SDS, and 1 mM EDTA supplemented with protease inhibitor cocktail from Roche (Sigma, St. Louis, MO) (Brown et al., 2011). The BCA kit (Abcam, ab102536) was used to determine the protein concentration. Protein (100 μg) from both control and treated samples was subjected to 4%–20% SDS‐PAGE. The resolved proteins in the gel were transferred to a PVDF membrane electrophoretically. The membrane was separately incubated first with Anti‐SREBP2 antibody (ab30682), Anti‐SCAP antibody (ab190103), Anti‐SP1 antibody (ab227383), Anti‐S1P antibody (ab140592), Anti‐S2P antibody (ab140594), Anti‐GAPDH antibody (ab181602) from Abcam (1:1,000) and then with goat anti‐rabbit secondary antibody (1:10,000) conjugated with horseradish peroxidase followed by ECL detection from Bio‐Rad (Hercules, CA).

Binding of SP1 to miR-200b-3p promoter was examined by ChIP assay with SimpleChIP Enzymatic Chromatin IP kit (CST). Transfected cells were crosslinked with 1% formaldehyde. Cross-linked chromatin was broken into 200 to 1000-bp fragments by ultrasonic. Chromatin was immunoprecipitated with SP1 (ab227383, Abcam) or IgG (#2729, CST) antibodies at 4 °C for 2 h. Immunoprecipitate complexes were collected with protein A/G-Sepharose beads (Roche, Basel, Switzerland). After washing, the enrichment was examined using PCR.

ChIP was implemented based on the previous study [19 (link)] with the rabbit IgG antibody (ab109489, 1: 300, Abcam) and the SP1-specific antibody (ab227383, 1: 200, Abcam). The enrichment of SP1 in the TUG1 promoter was detected using qPCR.