Mouse anti ub antibody

Immunoprecipitation experiment was performed to analyze the protein interactions or indicated PTMs as previously described (20 (link)). In brief, cell lysates were prepared in RIPA buffer and precipitated using anti–DDDDK-tag mAb-Magnetic Beads (MBL) or anti-GFP mAb-Magnetic Beads (MBL). Precipitated products were analyzed by Western blot using indicated antibodies. For the ubiquitination assay, cells were treated with MG132 (20 μmol/L) for 6 hours before collecting. The targeting protein ubiquitination was detected by mouse anti-Ub antibody (Santa Cruz Biotechnology).

Western blot was conducted to test the protein levels in mouse ventricular tissue and neonatal cardiomyocytes. We take the 2 mm area around the infarct of the pathological mouse heart as the peri-infarcted region. After quantification with the BCA Protein Assay kit (Beyotime, Shanghai, China), an equal amount of 60 μg protein from each group was fractionated by 8% SDS-PAGE and electro-blotted onto NC membranes (Pall, New York, USA). Membranes were incubated with primary antibodies overnight at 4 °C, including rabbit anti-Meis1 antibody (Abcam, UK, Cat#: ab19867, 1:1000), rabbit anti-Na_v1.5 antibody (Alomane laboratory, Israel, Cat#: ASC-005, 1:200), mouse anti-CDC20 antibody (Proteintech Group, America, Cat#: 10252-1-AP, 1:500), mouse anti-Ub antibody (Santa Cruz Biotechnology, Europe, Cat#: sc-8017, 1:1000), mouse anti-His antibody (Santa Cruz Biotechnology, Europe, Cat#: sc-57598, 1:200) and mouse anti-flag antibody (Sigma, USA, Cat#: F1804, 1:1000). Mouse anti-β-Actin antibody (Zhongshanjinqiao, Beijing, China, Cat#: TA-09, 1:1000) was used as an internal reference. The Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebraska, USA) was used for image capture and Image studio software was used to quantify bands in each group.