Immunoprecipitation kit

Co-IPs were performed using Immunoprecipitation kit (Abcam; catalog no.: ab206996) following the manufacturer’s instructions. Cells were washed once with PBS and incubated with 2 mM dithiobis(succinimidyl propionate) (Thermo Fisher Scientific; catalog no.: PG82081) for 30 min for reversible crosslinking. The crosslinking reaction was stopped by incubating the cells with 25 mM Tris for 15 min. Cells were then washed with PBS and lysed with 500 μl cold nondenaturing lysis buffer (Abcam; Immunoprecipitation kit; abID: ab206996). Cells we harvested were transferred to microcentrifuge tubes and incubated at 4 °C for 30 min on a rotatory mixer. After protein quantification, 500 μg of proteins were incubated with a primary antibody overnight at 4 °C on a rotatory mixer. Then, 25 μl of protein A/G Sepharose beads were added to the protein–antibody mix and incubated for 2 h at 4 °C. Beads were then collected and washed by slow speed centrifugations. Bound proteins were eluted in 40 μl 2× SDS-PAGE loading buffer (125 mM Tris–HCl [pH 6.8], 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.005% bromophenol blue) and loaded on SDS-PAGE. For IP from mouse brain, hippocampus tissue was lysed with ice-cold nondenaturing lysis buffer, supplemented with protease inhibitor, and co-IP was performed as discussed previously.

Immunoprecipitation of CFP1 protein using CFP1 antibodies was performed with the manufacturer’s instructions using immunoprecipitation kit (Abcam). In addition, CFP1 Western blotting following immunoprecipitation was performed to evaluate the specificity of CFP1 antibodies used for CFP1 ChIP-seq for Cfp1^f/f uterine samples (Supplementary Fig. 10). On Day 4, uterine horns of Cfp1^f/f and Cfp1^d/d mice were cut vertically, and epithelial and stromal cells were separated from the smooth muscles (n = 4 to 5 per each group). Then, tissues were incubated in a lysis buffer with protease inhibitors and mixed on a rocker at 4 °C for 1 h. The tissue extracts were transferred to a fresh tube, and a predetermined amount of antibodies was added on a rocker at 4 °C for 4 h. After antibody binding, protein A/G Sepharose beads were added on a rocker at 4 °C for 1 h. Next, protein A/G Sepharose beads were collected, washed, and eluted. The protein of interest was purified by low-speed centrifugation at 4 °C and used for Western blotting.

Specific immunoprecipitation of threonine phosphorylated proteins was performed using the immunoprecipitation kit from Abcam (ab206996, Cambridge, United Kingdom). The lysates used were the same as those described above. The protocol was scrupulously followed with the adaptations described by Wang et al. [43 (link)]. For 400 μg of proteins, 30 μg of specific threonine phosphorylated antibody (13–9200, Thermofisher, Waltham, MA, USA) was mixed with 30 μl of A/G sepharose beads. Separation of the proteins, antibody and beads was performed by adding the SDS loading buffer (Laemmli buffer, Bio-Rad, Hercules, CA, USA). The immunoprecipitated proteins were used directly in electrophoresis gel.