Sc 7962

Total proteins were extracted from T-HESCs using a lysis buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (1:100 dilution; CWBio, Beijing, China). All extracted proteins were heated at 100°C for 10 min and then stored at -80°C. The proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis system and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 and incubated with the following primary antibodies: anti-RARα (1:1,000 dilution; 62294, lot #1, Cell Signaling Technology, Danvers, MA, USA), anti-C/EBPβ (1:150 dilution; SC-7962, lot#12017, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-GAPDH (1:5,000 dilution; SA00001-1/SA00001-2, lot#20000275/20000311, Proteintech, Wuhan, China). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution, Proteintech). Western blotting was then performed, and the labeled protein bands were developed using HRP (Millipore). The intensity of the bands was determined using Image Lab software (Bio-Rad, Hercules, CA, USA).

Details of the specific antibodies that were used in the study are as follows: anti-CEP128 (HPA001116, Sigma-Aldrich, WB: 1:1000, IF: 1:200); anti-Ac-Tubulin (ab24610, abcam, IF: 1:1000); anti-NIN (A8215, ABclonal, WB: 1:1000); anti-CEP170 (27325-1-AP, Proteintch, WB: 1:500); anti-YY1 (sc-7341, Santa Cruz Biotechnology, WB: 1:500); anti-GR-β (sc-393232, Santa Cruz Biotechnology, WB: 1:500); anti-SEPT4 (Septin 4) (A10238, ABclonal, IF: 1:100); anti-RCBTB2 (13225-1-AP, Proteintech, WB: 1:1000, IF: 1:50); anti-WBP2NL (22587-1-AP, Proteintech, WB: 1:500, IF: 1:50); anti-CRISP1(MAB4675, R&D Systems, WB: 1:500, IF: 1:50); anti-PRSS55 (bs-19443R, Bioss, WB: 1:1000, IF: 1:50); anti-ubiquitin (ab7780, Abcam, WB: 1:500), anti-GAPDH (ab8245, Abcam, WB: 1:1000); anti-CEBPβ (sc-7962, Santa Cruz Biotechnology, WB: 1:500); anti-Centriolin (sc-365521, Santa Cruz Biotechnology, WB: 1:500).

Chromatin immunoprecipitation (ChIP) was performed following the instructions for SimpleChIP^® Enzymatic Chromatin IP Kit (Cell Signaling). For each immunoprecipitation performed, 1.6 × 10⁶ 3T3-L1 cells were seeded and grown to 2 days post–confluence (T0) before being treated, or not, with 1.0 uM DEX. After 20 h cells were fixed with 1% formaldehyde followed by glycine incubation. After cell lysis, chromatin was fragmented by micrococcal digestion and nuclear membranes were broken by sonication with a Bioruptor Sonicator (Diagenode). An aliquot of the digested chromatin (Input) was removed before sample immunoprecipitation with antibodies against either CEBPB (sc-7962, Santa Cruz Biotechnologies,) or a non-specific IgG control (Normal Rabbit IgG #2729, Cell Signaling). The complex co-precipitates were captured by Protein G magnetic beads and the cross-links were reverted before DNA purification. Chip-qPCR primers are listed in Table 1.

To investigate protein association, immunoprecipitations were performed with Protein G sepharose (Sigma) following the manufacturer’s instruction. Briefly, ARCMs were lysed with immunoprecipitation buffer (50 mM Tris, 250 mM NaCl, 0.25% v/v TritonX- 100, and 10% Glycerol, pH7.4). After, quantification, 2 mg of the protein extract was immunoprecipitated with antibodies against CEBPβ (sc-7962, Santa Cruz Biotechnology) or IgG (Cell Signaling, 2729). Immune complexes were eluted in Laemmli sample buffer (65.8 mM Tris-HCl, pH6.8, 2.1% SDS, 26.3% glycerol, 0.01% bromophenol blue). Precipitated and input proteins were subjected to SDS-PAGE and immunoblotted using the respective antibodies.