Journal: Clinical Proteomics

Article Title: Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS

doi: 10.1186/s12014-022-09391-9

Figure Lengend Snippet: Comparisons of differentially expressed proteins and two protein modifications between EXP and CON groups. EXP: The experimental group with sperm DNA fragmentation index (DFI) ≥ 30%; CON: The control group with sperm DFI < 30%; n = 2 for each group. A Western blot showed that the expression levels of RAD23B and DFFA in the experimental group were significantly higher than those in the control group, which were consistent with the results of proteomic analysis. B Sperm proteins were separated by 12% of polyacrylamide gel electrophoresis and stained by Coomassie brilliant blue, and there was no obvious changes in sperm protein levels between the experimental group and control group. C Sperm proteins were separated by 12% of polyacrylamide gel electrophoresis and detected with antibodies against ubiquitins, and the results showed that the ubiquitination modification of sperm proteins at approximately 100 kD in the experimental group was significantly higher than that in the control group. D Sperm proteins were separated by 12% of polyacrylamide gel electrophoresis and detected with antibodies against acetyllysine, and the results showed that the acetylation modification of sperm proteins at 20–45 kD in the experimental group was significantly higher than that in the control group

Article Snippet: 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.

Techniques: Control, Western Blot, Expressing, Polyacrylamide Gel Electrophoresis, Staining, Ubiquitin Proteomics, Modification

Journal: Clinical proteomics

Article Title: Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS.

doi: 10.1186/s12014-022-09391-9

Figure Lengend Snippet: Fig. 5 Comparisons of differentially expressed proteins and two protein modifications between EXP and CON groups. EXP: The experimental group with sperm DNA fragmentation index (DFI) ≥ 30%; CON: The control group with sperm DFI < 30%; n = 2 for each group. A Western blot showed that the expression levels of RAD23B and DFFA in the experimental group were significantly higher than those in the control group, which were consistent with the results of proteomic analysis. B Sperm proteins were separated by 12% of polyacrylamide gel electrophoresis and stained by Coomassie brilliant blue, and there was no obvious changes in sperm protein levels between the experimental group and control group. C Sperm proteins were separated by 12% of polyacrylamide gel electrophoresis and detected with antibodies against ubiquitins, and the results showed that the ubiquitination modification of sperm proteins at approximately 100 kD in the experimental group was significantly higher than that in the control group. D Sperm proteins were separated by 12% of polyacrylamide gel electrophoresis and detected with antibodies against acetyllysine, and the results showed that the acetylation modification of sperm proteins at 20–45 kD in the experimental group was significantly higher than that in the control group

Article Snippet: 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.

Techniques: Control, Western Blot, Expressing, Polyacrylamide Gel Electrophoresis, Staining, Ubiquitin Proteomics, Modification

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: miR-210 attenuates the hypoxia-induced increase in DNA fragmentation through the inhibition of GSK3β kinase activity. ( A ) Quantitative TUNEL assay determining apoptotic DNA fragmentation, expressed as experimental blank-corrected absorbances (O.D) measured at λ 450 (450 nm). ( B ) Qualitative Western blot depicting the processing of the DFF45 into cleaved -DFF45 (p11 fragment) as a surrogate measure of caspase-3-induced DFF40 endonuclease activity. ( C ) Quantitative ELISA determining the levels of cleaved -DFF45 (p11 fragment), expressed as experimental blank-corrected absorbance (O.D) measured at λ 570 (570 nm). ( D ) Quantitative DFF40 endonuclease activity assay, expressed as experimental blank-corrected absorbances (O.D) measured at λ 405 (405 nm). miR-210 expression levels in the respective cell lysates were determined by the miR-210 hybridization immunoassay (as described in ) and are reported in . The validation of the ectopic expression of the HA-tagged ca -GSK3β-S9A mutant in the pertinent experimental groups was performed by ELISA immunoassay and is reported in ). GSK3β kinase activity (as described in ) was measured in all experimental groups to corroborate and validate the translative effects of the ectopic expression of the ca -GSK3β-S9A mutant (reported in ). All data are expressed as mean ± S.D from three technical replicates for each of the four biological replicates belonging to each experimental group ( n = 4). ** p ≤ 0.01; **** p ≤ 0.0001; ns: not significant ( p > 0.05). OE: miR-210 overexpression; O.D: optical density; S.D: standard deviation.

Article Snippet: cleaved -DFF45 (Asp 224 ) , WB 1:500 , 5 μg , Rabbit , Cell Signaling Technology , 9731 , AB_329954.

Techniques: Inhibition, Activity Assay, TUNEL Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Hybridization, Biomarker Discovery, Mutagenesis, Over Expression, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: List of antibodies and antibody-blocking peptides used in the study.

Article Snippet: cleaved -DFF45 (Asp 224 ) , WB 1:500 , 5 μg , Rabbit , Cell Signaling Technology , 9731 , AB_329954.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Peptide ELISA, Activity Assay