Anti zbtb16

Ventricles from PND1 WT and cMyBP-C^-/- mouse hearts were isolated and homogenate was fractionated to enrich for nuclear proteins using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, cat #78833) according to manufacturer instructions. Total protein in nuclear fractions was quantified using a standard BCA protein assay (Thermo Fisher) using BSA protein standards. 25 μg of total protein homogenate was fractionated in 4–20% SDS-PAGE gels (Bio-Rad) and transferred overnight at 15 V in Tris–glycine buffer. Immunoblotting was performed for Zbtb16 (anti-Zbtb16; 1:1,000, Abcam, ab189849) and the nuclear loading control Histone 3 (anti-H3; 1:1,000, Cell Signaling cat# 9715), as described above for Xirp2.

The bone of NCs and OP patients and the calvarial and femoral bones of mice were collected for immunofluorescence analysis. Bone tissue sections were deparaffinized and rehydrated. Citrate buffer (pH 6.0) at a concentration of 10 mmol·L⁻¹ was used for antigen retrieval. Sections were immersed in citrate buffer and microwaved for 15 min. Cultured MSCs were fixed with 4% PFA for 15 min before immunofluorescence. After permeabilization with 0.5% Triton X-100 for 20 min, bone sections or MSCs were blocked with 10% FBS in PBS for 1 h. After incubation with anti-ZBTB16 (Abcam, Cat. No. ab104854), anti-BRD4 (Cell Signaling Technology, Cat. No. 13440S), anti-POL II CTD (Santa Cruz, Cat. No. sc-47701) or anti-RPAP2 (Proteintech, Cat. No. 17401-1-AP) primary antibodies overnight at 4 °C, the samples were incubated with the following secondary antibodies for 1 h at room temperature: anti-mouse Alexa 488 (Cell Signaling Technology, Cat. No. 4408) and anti-rabbit Alexa 555 (Cell Signaling Technology, Cat. No. 4413). We used DAPI antifade mounting medium (Beyotime, Cat. No. P0131) for mounting. Images were captured using a Zeiss LSM 880 confocal microscope.