Journal: Chinese Medical Journal

Article Title: SENP2-mediated SERCA2a deSUMOylation increases calcium overload in cardiomyocytes to aggravate myocardial ischemia/reperfusion injury

doi: 10.1097/CM9.0000000000002757

Figure Lengend Snippet: The primers of mouse Sumo1 , Ubc9 , Senp1 , and Senp2 .

Article Snippet: The membranes were immunoblotted overnight at 4°C with primary antibodies against Sumo1, UBC9, Senp1 (c-12) (1:1000; Santa Cruz Biotechnology, Inc., CA, USA), Senp2, Serca2a (1:1000; Abcam, Cambridge, UK), and β-actin (1:5000; Beijing Zhongshan Golden bridge Biotechnology Co., Beijing, China).

Techniques:

Journal: Chinese Medical Journal

Article Title: SENP2-mediated SERCA2a deSUMOylation increases calcium overload in cardiomyocytes to aggravate myocardial ischemia/reperfusion injury

doi: 10.1097/CM9.0000000000002757

Figure Lengend Snippet: Detection of Sumo1, Ubc9, Senp1, and Senp2 mRNA and proteins in C57BL/6J mice at time of 0 h, 2 h, 6 h, 12 h, and 3 days after I/RI ( n = 3). (A) RT-qPCR detection of Sumo1, Ubc9, Senp1, and Senp2 mRNA at different time points. (B) Western blotting detection of Sumo1, Ubc9, Senp1, and Senp2 proteins at different time points. I/RI: Ischemia/reperfusion injury; mRNA: messenger RNA; RT-qPCR: Quantitative real-time polymerase chain reaction; Senp1/2: sentrin/small ubiquitin-related modifier (SUMO)-specific protease 1/2; Ubc9:Ubiquitin-conjugating enzyme 9. * Indicates comparison with the sham group, * P <0.050, ** P <0.010, and *** P <0.001. † Indicates comparison with the I/RI 0 h group, † P <0.050, †† P <0.010, and ††† P <0.001. ‡ Indicates comparison with the I/RI 2 h group, ‡ P <0.050, ‡‡ P <0.010, and ‡‡‡ P <0.001. § Indicates comparison with the I/RI 6 h group, § P <0.050, §§ P <0.010, and §§§ P <0.001. || Indicates comparison with the I/RI 12 h group, || P <0.050, |||| P <0.010, and |||||| P <0.001.

Article Snippet: The membranes were immunoblotted overnight at 4°C with primary antibodies against Sumo1, UBC9, Senp1 (c-12) (1:1000; Santa Cruz Biotechnology, Inc., CA, USA), Senp2, Serca2a (1:1000; Abcam, Cambridge, UK), and β-actin (1:5000; Beijing Zhongshan Golden bridge Biotechnology Co., Beijing, China).

Techniques: Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Comparison

Journal: Chinese Medical Journal

Article Title: SENP2-mediated SERCA2a deSUMOylation increases calcium overload in cardiomyocytes to aggravate myocardial ischemia/reperfusion injury

doi: 10.1097/CM9.0000000000002757

Figure Lengend Snippet: Expression of Sumo1, Ubc9, Senp1, and Senp2 mRNA and proteins in cardiomyocytes and HL-1 cells at time 0 h, 2 h, 6 h, 12 h, and 3 days after sI/RI ( n = 3). (A) Morphology of cardiomyocytes isolated from C57BL/6J mice before and after I/RI (Scale bar=100 μm). (B) RT-qPCR detection of Sumo1, Ubc9, Senp1, and Senp2 mRNA in cardiomyocytes. (C) Western blot detection of Sumo1, Ubc9, Senp1, and Senp2 proteins in cardiomyocytes. (D) Morphology of HL-1 (Scale bar=100 μm). (E) RT-qPCR detection of Sumo1, Ubc9, Senp1, and Senp2 mRNA in HL-1 cells. (F) Western blot detection of Sumo1, Ubc9, Senp1, and Senp2 proteins in HL-1 cells. CON: Control; I/RI: Ischemia/reperfusion injury; RT-qPCR: Quantitative real-time polymerase chain reaction; Senp1/2: sentrin/small ubiquitin-related modifier (SUMO)-specific protease 1/2; sI/RI: simulation I/RI; Ubc9:Ubiquitin-conjugating enzyme 9. * Indicates comparison with the sham group, * P <0.050, ** P <0.010, and *** P <0.001. † Indicates comparison with the I/RI 0 h group, † P <0.050, †† P <0.010, and ††† P <0.001. ‡ Indicates comparison with the I/RI 2 h group, ‡ P <0.050, ‡‡ P <0.010, and ‡‡‡ P <0.001. § Indicates comparison with the I/RI 6 h group, § P <0.050, §§ P <0.010, and §§§ P <0.001. || Indicates comparison with the I/RI 12 h group, || P <0.050, |||| P <0.010, and |||||| P <0.001.

Article Snippet: The membranes were immunoblotted overnight at 4°C with primary antibodies against Sumo1, UBC9, Senp1 (c-12) (1:1000; Santa Cruz Biotechnology, Inc., CA, USA), Senp2, Serca2a (1:1000; Abcam, Cambridge, UK), and β-actin (1:5000; Beijing Zhongshan Golden bridge Biotechnology Co., Beijing, China).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Control, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Comparison

Journal: Chinese Medical Journal

Article Title: SENP2-mediated SERCA2a deSUMOylation increases calcium overload in cardiomyocytes to aggravate myocardial ischemia/reperfusion injury

doi: 10.1097/CM9.0000000000002757

Figure Lengend Snippet: Localization of Senp1, Senp2, and Serca2a in cardiomyocytes using immunofluorescence and influence of I/RI on localization (Scale bar=10 μm; n = 3). (A) Bright-field image of cardiomyocytes. (B) DAPI staining of cardiomyocytes (blue). (C) Serca2a protein staining with Alexa Fluor ® 568 (orange). (D) Senp1 protein staining with Alexa Fluor ® 647 (red). (E) Senp2 protein staining with Alexa Fluor ® 488 (green). (F) Co-localization of Senp1 and Serca2a. (G) Co-localization of Senp2 and Serca2a. (H) Magnification of Senp1 and Senp2 location. (I) Localization of Serca2a and Senp1 proteins in cardiomyocytes 6 h after I/RI. (J) Localization of Serca2a and Senp2 proteins in cardiomyocytes 6 h after I/RI. Con: Control; DAPI: 4′,6-Diamidino-2′-phenylindole; I/RI: Ischemia/reperfusion injury; Senp1/2: sentrin/small ubiquitin-related modifier (SUMO)-specific protease 1/2; Ubc9:Ubiquitin-conjugating enzyme 9.

Article Snippet: The membranes were immunoblotted overnight at 4°C with primary antibodies against Sumo1, UBC9, Senp1 (c-12) (1:1000; Santa Cruz Biotechnology, Inc., CA, USA), Senp2, Serca2a (1:1000; Abcam, Cambridge, UK), and β-actin (1:5000; Beijing Zhongshan Golden bridge Biotechnology Co., Beijing, China).

Techniques: Immunofluorescence, Staining, Control, Ubiquitin Proteomics

Journal: Frontiers in Immunology

Article Title: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms

doi: 10.3389/fimmu.2018.01142

Figure Lengend Snippet: Confirmation of differentially expressed proteins and phosphoproteins by western blotting and Phos-tag western Blotting. (A) Analysis of ubiquitin conjugating enzyme E2 I, ubiquitin conjugating enzyme E2 L3, glyceraldehyde-3-phosphate dehydrogenase, β-actin expression levels in foot-and-mouth disease virus (FMDV)-infected and control cells by western blotting. SILAC-ratios and immunoblotting ratios (infection/control) were shown on the right side. (B) Analysis of the dynamic phosphorylation alterations of the three differentially phosphoproteins (ribosomal protein L15, chromosome 5 open reading frame 24, and FOS-like 2) in FMDV-infected and control cells by Phos-tag western blotting.

Article Snippet: To confirm the expression levels of GAPDH, ubiquitin conjugating enzyme E2 I (UBE2I), ubiquitin conjugating enzyme E2 L3 (UBE2L3), ribosomal protein L15 (RPL15), chromosome 5 open reading frame 24 (C5ORF24) and FOS-like 2 (FOSL2), anti-GAPDH antibody (Beyotime, China), anti-UBE2I antibody (Proteintech, China), anti-UBE2L3 antibody (Proteintech, China), anti-RPL15 antibody (Proteintech, China), anti-C5ORF24 antibody (Proteintech, China), and anti-FOSL2 antibody (Proteintech, China) were used for immunoblotting.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Virus, Infection, Control, Multiplex sample analysis, Phospho-proteomics