Ne per protein extraction kit

Detailed methods for co-IP have been described previously.⁴⁹ (link)^,50 (link) The IFITM4P-binding proteins were studied by RNA pull-down assays using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Biotinylated IFITM4P and antisense sequences were synthesized using a TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific). The cytoplasmic fraction obtained using an NE-PER Protein Extraction Kit (Thermo Fisher Scientific) was incubated overnight with biotinylated with IFITM4P, followed by precipitation with streptavidin magnetic beads. The retrieved protein was eluted from the RNA-protein complex and analyzed by immunoblotting or silver staining. Silver staining was performed using a silver staining kit (Beyotime, China) according to the manufacturer's instructions. Ubiquitinating sites were studied via MS (Novogene Bioinformatics Technology, Beijing, China).
The RIP assays were performed using an EZ-Magna RIP kit (Millipore). Leuk-1 cells (4 × 10⁸) were lysed using a complete RIP lysis buffer. The lysates were immunoprecipitated in RIP buffer with anti-HuR antibody-conjugated magnetic beads (Abcam, Cambridge, UK), SASH1 antibody, or IgG. The precipitated RNAs were analyzed by qRT-PCR. Mouse IgG and HuR RNA were used as negative and positive controls, respectively.

Subcellular fractionation and DNA binding assays were conducted. Nuclear and cytoplasmic protein of OS cells was extracted using an NE-PER protein extraction kit (Thermo Scientific, USA) according to the manufacturer's instructions. For western blot analysis, tubulin and histone deacetylase 1 (HDAC1) were used for the cytoplasmic and nuclear loading control, respectively.
Oligonucleotide pull-down assays were performed with an annealed nucleotide comprising the STAT1 consensus site (5′-CATGTTATGCATATTCCTGTAAGTG-3′) and STAT3 consensus site (5'-GATCCTTCTGGGAATTCCTAGATC-3') with a 5-biotin label. Nuclear extracts (50-100 μg) were incubated for 1 h at 4 °C with 1 μg oligonucleotide in binding buffer, and then Sepharose-streptavidin (50 μL; Sigma) was added for 2 h at 4 °C. After three washes in PBS, the complexes were suspended in SDS sample buffer and processed for western blotting, as described above, and probed with anti-STAT1, STAT3 antibodies (CST).

Nuclear and cytoplasmic proteins of cells were extracted using the NE-PER Protein Extraction Kit (#78833, Thermo Scientific, USA) according to the manufacturer's protocol. For western blotting analyses, histone deacetylase 1 (HDAC1, Santa Cruz biotechnology Inc. USA) were used as marker for nuclear fraction.

WB was carried out as described previously46 (link)47 (link)49 . In brief, MCF7 cells grown in six-well tissue culture plates in medium supplemented with CD-FBS for 48 h were treated without (EtOH, 0.01%) or with 10⁻⁹ M E2 and/or 10⁻⁷ ICI for 3, 6 or 24 h. At the termination, cells were collected and protein isolation was performed using NE-PER protein extraction kit (Thermo-Fisher). Protein content in extracts was measured with Bradford Protein Assay (Bio-Rad). Nuclear extracts (25 μg or 100 μg) were then subjected to SDS 10%-PAGE. Proteins were probed with an antibody specific to CXXC5 (ab106533, Abcam) or Flag (Sigma-Aldrich) followed by a secondary antibody conjugated with the horseradish peroxidase (Santa Cruz). Protein images were developed using the ECL-Plus Western Blotting kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and captured with ChemiDoc™ Imaging System (Bio-Rad). Precision Plus Protein™ Dual Color Standards (Bio-Rad) was used as molecular marker in WB. The quantification of images was carried out using ImageJ image processing program (https://imagej.nih.gov/ij/).