Ptm 1106

Co-immunoprecipitation was performed as follows. The tissue extracts or cell lysates were immunoprecipitated with 3 μg of anti-OTUD7B (rabbit polyclonal, 16605-1-AP, Proteintech), anti-KLF4 (rabbit polyclonal, ab106629, Abcam), anti-NMHC IIA (rabbit polyclonal, 11128-1-AP, Proteintech) or anti-ubiquitin (rabbit polyclonal, PTM-1106, PTM BIO), respectively, for 1 h at 4 °C, then incubated with protein A-agarose overnight at 4 °C. The complexes of Protein A-agarose-antibody were collected by centrifugation at 12,000 rpm for 2 min at 4 °C, and washed 4 times with 500 μl of IPH buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, and 0.1 mM phenylmethylsulfonyl fluoride) for 20 min at 4 °C. The bound proteins were resolved using SDS-polyacrylamide gel followed by Western blotting using anti-OTUD7B (rabbit polyclonal, 1:1000, 16605-1-AP, Proteintech), anti-KLF4 (rabbit polyclonal, 1:1000, ab106629, Abcam), anti-NMHC IIA (rabbit polyclonal, 1:1000, 11128-1-AP, Proteintech) or anti-ubiquitin (rabbit polyclonal, 1:2000, PTM-1106, PTM BIO) antibodies.

The reliability of proteomics could be validated by Western blotting. Two differentially expressed proteins (DFFA and RAD23B) and two major protein modifications (ubiquitination and acetylation) were selected for Western blotting.
Briefly, 20 µg of protein was separated by 12% polyacrylamide gel electrophoresis (concentrated gel at 80 V for 50 min and separated gel at 120 V for 2 h). Subsequently, the gels were stained with Coomassie blue dye or proteins were transferred to polyvinyl difluoride (PVDF) membranes (Millipore, Bedford, Mass, USA) at 30 V overnight at 4 °C using the Mini TransBlot transfer unit (Bio-Rad, Hercules, CA, USA). Then, the membranes were blocked in tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% nonfat dry milk for 60 min at room temperature, and incubated with antibodies against acetyllysine (PTM-101, 1:1000, PTM BIO, Hangzhou, China), ubiquitins (PTM-1106, 1:1000, PTM BIO, Hangzhou, China), DFFA (ab108924, Abcam, USA) and RAD23B (12,121–1-AP, Proteintech, China) overnight at 4 °C, respectively. Subsequently, the membranes were washed with PBST (PBS, 0.05% Tween-20), and incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000; Pierce, Rockford, IL, USA) for 1 h at room temperature. Last, the membranes were detected by the enhanced chemiluminescence.