Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: The generation of Mkrn2 KO and WT Mice

Article Snippet: For immunohistochemical analysis, anti-MKRN2 antibody (catalog# NB100-55250, Novus Biologicals, Inc., Littleton, CO, UK) was used at a 1:2000 dilution, anti-cleaved caspase 3 antibody (catalog# 19677-1-AP, Proteintech, Wuhan, China) was used at a 1:200 dilution, anti-BCL2 Associated X (BAX) antibody (catalog# 50599-2-Ig, Proteintech) was used at a 1:100 dilution, anti-p53 apoptosis effector related to PMP22 (PERP) antibody (catalog# AB_48032, Abcam, Inc., London, UK) was used at a 1:100 dilution, and anti-p53 (DO-7) antibody (catalog# 48818, CST, Inc., Boston, MA, USA) was used at a 1:100 dilution.

Techniques:

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: Primers used in this study

Article Snippet: For immunohistochemical analysis, anti-MKRN2 antibody (catalog# NB100-55250, Novus Biologicals, Inc., Littleton, CO, UK) was used at a 1:2000 dilution, anti-cleaved caspase 3 antibody (catalog# 19677-1-AP, Proteintech, Wuhan, China) was used at a 1:200 dilution, anti-BCL2 Associated X (BAX) antibody (catalog# 50599-2-Ig, Proteintech) was used at a 1:100 dilution, anti-p53 apoptosis effector related to PMP22 (PERP) antibody (catalog# AB_48032, Abcam, Inc., London, UK) was used at a 1:100 dilution, and anti-p53 (DO-7) antibody (catalog# 48818, CST, Inc., Boston, MA, USA) was used at a 1:100 dilution.

Techniques: Sequencing

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: The sets of DEGs enriched in Mkrn 2-KO were compared to those from the Mkrn 2-WT mice. ( a ) Volcano plot of DEGs in MEFs isolated from Mkrn 2-KO and Mkrn 2-WT mice. DEGs are color coded according to whether they are up- or down-regulated (logFC >1 or <−1, P < 0.05); 614 genes upregulated in Mkrn 2-KO group, red; 754 genes downregulated in Mkrn 2-KO group, blue. ( b ) Scatter plot illustrating relative gene expression levels by group, with color coding as in a. ( c ) Biological pathways represented among the identified DEGs, as determined by KEGG analysis. DEG: differential expression gene; Mkrn 2: makorin-2; KO: knockout; WT: wild type; MEF: mouse embryonic fibroblast; KEGG: Kyoto Encyclopedia of Genes and Genomes; logFC: log fold change; mTOR: Mechanistic Target Of Rapamycin Kinase.

Article Snippet: For immunohistochemical analysis, anti-MKRN2 antibody (catalog# NB100-55250, Novus Biologicals, Inc., Littleton, CO, UK) was used at a 1:2000 dilution, anti-cleaved caspase 3 antibody (catalog# 19677-1-AP, Proteintech, Wuhan, China) was used at a 1:200 dilution, anti-BCL2 Associated X (BAX) antibody (catalog# 50599-2-Ig, Proteintech) was used at a 1:100 dilution, anti-p53 apoptosis effector related to PMP22 (PERP) antibody (catalog# AB_48032, Abcam, Inc., London, UK) was used at a 1:100 dilution, and anti-p53 (DO-7) antibody (catalog# 48818, CST, Inc., Boston, MA, USA) was used at a 1:100 dilution.

Techniques: Isolation, Gene Expression, Quantitative Proteomics, Knock-Out

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: DEGs and PERP expression levels in Mkrn 2-KO cells and tissues. ( a ) Expression levels of DEGs for the Mkrn 2-KO and Mkrn 2-WT MEFs are analyzed by qRT-PCR. Data are presented and analyzed by the Student's t -test from three independent experiments performed in triplicate. ** Significant difference compared with WT group ( P < 0.01). 293T cells were co-transfected with an Mkrn2 shRNA (shMkrn2) or negative control (Mock) vector plus an MKRN2 expression vector or control vector. ( b ) qRT-PCR is used to analyze the mRNA expression of PERP . Data are presented as mean ± s.d. from three independent experiments performed in triplicate. ( c ) Western blot is used to analyze the protein expression of PERP, densitometric analysis of Western blot signals (right). ( b and c ) Results are presented and analyzed by the two-way ANOVA, *Significant difference at P < 0.05, **Significant difference at P < 0.01. ( d ) Correlation between MKRN 2 and PERP expression levels in GEO dataset GSE6872 (contains sperm samples), as analyzed by Pearson's correlation analysis. DEG: differential expression gene; Mkrn 2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to peripheral myelin protein 22 (PMP22); qRT-PCR: quantitative real-time polymerase chain reaction; s.d.: standard deviation; MEF: mouse embryonic fibroblast; GEO: Gene Expression Omnibus.

Article Snippet: For immunohistochemical analysis, anti-MKRN2 antibody (catalog# NB100-55250, Novus Biologicals, Inc., Littleton, CO, UK) was used at a 1:2000 dilution, anti-cleaved caspase 3 antibody (catalog# 19677-1-AP, Proteintech, Wuhan, China) was used at a 1:200 dilution, anti-BCL2 Associated X (BAX) antibody (catalog# 50599-2-Ig, Proteintech) was used at a 1:100 dilution, anti-p53 apoptosis effector related to PMP22 (PERP) antibody (catalog# AB_48032, Abcam, Inc., London, UK) was used at a 1:100 dilution, and anti-p53 (DO-7) antibody (catalog# 48818, CST, Inc., Boston, MA, USA) was used at a 1:100 dilution.

Techniques: Expressing, Quantitative RT-PCR, Transfection, shRNA, Negative Control, Plasmid Preparation, Control, Western Blot, Quantitative Proteomics, Knock-Out, Real-time Polymerase Chain Reaction, Standard Deviation, Gene Expression

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: Correlation of PERP levels with spermatogenesis. ( a ) Relative expression levels of the Mkrn 2 in normal and OAT sperm samples from the GSE6872 dataset. Data are presented and analyzed by two-tailed, unpaired Student's t -test. ** Significant differences compared with WT group ( P < 0.01). ( b ) Enrichment and ranking of genes in the PERP -high and PERP -low expression groups of the GSE6872 dataset are assessed by GSEA analysis (C5: GO gene sets; BP: GO biological process; gene set: male gamete generation) and the corresponding clustering heatmap of genes that contribute most to enrichment score is on the right. ( c ) Enrichment and ranking of genes in the PERP -high and PERP -low expression groups of the GSE6872 dataset are assessed by GSEA analysis (H: hallmark gene set; gene set: spermatogenesis) and the corresponding clustering heatmap of genes that contribute most to enrichment score is on the right ( PERP- low datasets: GSM158476, GSE158477, and GSM158479 and PERP -high datasets: GSM158478, GSM158480, GSM158481, GSM158482, and GSM158483). ( d ) Correlations between spermatogenic signature scores and PERP expression levels are determined by Spearman's rank correlation analysis. Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; GSEA: gene set enrichment analysis; OAT: oligoasthenoteratozoospermia.

Article Snippet: For immunohistochemical analysis, anti-MKRN2 antibody (catalog# NB100-55250, Novus Biologicals, Inc., Littleton, CO, UK) was used at a 1:2000 dilution, anti-cleaved caspase 3 antibody (catalog# 19677-1-AP, Proteintech, Wuhan, China) was used at a 1:200 dilution, anti-BCL2 Associated X (BAX) antibody (catalog# 50599-2-Ig, Proteintech) was used at a 1:100 dilution, anti-p53 apoptosis effector related to PMP22 (PERP) antibody (catalog# AB_48032, Abcam, Inc., London, UK) was used at a 1:100 dilution, and anti-p53 (DO-7) antibody (catalog# 48818, CST, Inc., Boston, MA, USA) was used at a 1:100 dilution.

Techniques: Expressing, Two Tailed Test, Knock-Out

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: Association of PERP and p53 expression levels in Mkrn 2-KO cells and tissues. ( a ) Expression levels of p53 are analyzed by GEO dataset GSE6872. Data are presented and analyzed by two-tailed, unpaired Student's t -test. * Significant difference compared with the normal group ( P < 0.05). ( b ) Expression levels of MKRN2, p53, and PERP in MEFs are analyzed by Western blot (left), densitometric analysis of Western blot signals (right). ( c ) Expression levels of p53 and PERP in MEFs transfected with a p53 vector or CON as indicated are analyzed by Western blot (left), densitometric analysis of Western blot signals (right). ( d ) PERP expression levels are determined by Western blot in 293T cells co-transfected with vector and control, Mkrn2 and CON, Mkrn2 and p53, respectively (left), densitometric analysis of Western blot signals (right). ( c and d ) Results are presented and analyzed by the two-way ANOVA from three independent experiments and each performed for three times. * Significant difference at P < 0.05, ** Significant difference at P < 0.01. CON: negative control vector; Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; GEO: Gene Expression Omnibus; MEF: mouse embryonic fibroblast.

Article Snippet: For immunohistochemical analysis, anti-MKRN2 antibody (catalog# NB100-55250, Novus Biologicals, Inc., Littleton, CO, UK) was used at a 1:2000 dilution, anti-cleaved caspase 3 antibody (catalog# 19677-1-AP, Proteintech, Wuhan, China) was used at a 1:200 dilution, anti-BCL2 Associated X (BAX) antibody (catalog# 50599-2-Ig, Proteintech) was used at a 1:100 dilution, anti-p53 apoptosis effector related to PMP22 (PERP) antibody (catalog# AB_48032, Abcam, Inc., London, UK) was used at a 1:100 dilution, and anti-p53 (DO-7) antibody (catalog# 48818, CST, Inc., Boston, MA, USA) was used at a 1:100 dilution.

Techniques: Expressing, Two Tailed Test, Western Blot, Transfection, Plasmid Preparation, Control, Negative Control, Knock-Out, Gene Expression

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: p53/PERP expression levels in Mkrn 2-KO mouse testes. ( a ) TUNEL assay to assess the levels of apoptosis in Mkrn 2 KO versus WT mouse testis. Immunohistochemical staining of ( b ) p53 and ( c ) PERP expression in the testes of Mkrn 2-KO and Mkrn 2-WT mice. Scale bars=50 μm. ( d ) Western blot assay to test expression levels of MKRN2, p53, and PERP (left); and the different densitometric levels between two groups: Mkrn 2-KO and Mkrn 2-WT mouse testes are analyzed by the two-tailed, unpaired Student's t -test to compare the data of the two groups from three independent experiments (right). ** Significant difference at P < 0.01. Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; MEF: mouse embryonic fibroblast; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: For immunohistochemical analysis, anti-MKRN2 antibody (catalog# NB100-55250, Novus Biologicals, Inc., Littleton, CO, UK) was used at a 1:2000 dilution, anti-cleaved caspase 3 antibody (catalog# 19677-1-AP, Proteintech, Wuhan, China) was used at a 1:200 dilution, anti-BCL2 Associated X (BAX) antibody (catalog# 50599-2-Ig, Proteintech) was used at a 1:100 dilution, anti-p53 apoptosis effector related to PMP22 (PERP) antibody (catalog# AB_48032, Abcam, Inc., London, UK) was used at a 1:100 dilution, and anti-p53 (DO-7) antibody (catalog# 48818, CST, Inc., Boston, MA, USA) was used at a 1:100 dilution.

Techniques: Expressing, TUNEL Assay, Immunohistochemical staining, Staining, Western Blot, Two Tailed Test, Knock-Out

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: The generation of Mkrn2 KO and WT Mice

Article Snippet: The tissues lysates were subjected to three sets of 15-s pulses between pulses for 15 min on ice and then centrifuged at 21 100 g at 4°C for 1 h. The extracts were subjected to immunoblot using antibodies for MKRN2 (1:2000 dilution; catalog# NB100-55250, Novus Biologicals), PERP (1:500 dilution; catalog# AB_48032, Abcam), p53 (DO-7) (1:1000 dilution; catalog# 48818, CST), and anti-p53 (acetyl-K120) (1:1000 dilution; catalog# HW-186, SAB, Inc., Nanjing, China) as described above.

Techniques:

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: Primers used in this study

Article Snippet: The tissues lysates were subjected to three sets of 15-s pulses between pulses for 15 min on ice and then centrifuged at 21 100 g at 4°C for 1 h. The extracts were subjected to immunoblot using antibodies for MKRN2 (1:2000 dilution; catalog# NB100-55250, Novus Biologicals), PERP (1:500 dilution; catalog# AB_48032, Abcam), p53 (DO-7) (1:1000 dilution; catalog# 48818, CST), and anti-p53 (acetyl-K120) (1:1000 dilution; catalog# HW-186, SAB, Inc., Nanjing, China) as described above.

Techniques: Sequencing

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: The sets of DEGs enriched in Mkrn 2-KO were compared to those from the Mkrn 2-WT mice. ( a ) Volcano plot of DEGs in MEFs isolated from Mkrn 2-KO and Mkrn 2-WT mice. DEGs are color coded according to whether they are up- or down-regulated (logFC >1 or <−1, P < 0.05); 614 genes upregulated in Mkrn 2-KO group, red; 754 genes downregulated in Mkrn 2-KO group, blue. ( b ) Scatter plot illustrating relative gene expression levels by group, with color coding as in a. ( c ) Biological pathways represented among the identified DEGs, as determined by KEGG analysis. DEG: differential expression gene; Mkrn 2: makorin-2; KO: knockout; WT: wild type; MEF: mouse embryonic fibroblast; KEGG: Kyoto Encyclopedia of Genes and Genomes; logFC: log fold change; mTOR: Mechanistic Target Of Rapamycin Kinase.

Article Snippet: The tissues lysates were subjected to three sets of 15-s pulses between pulses for 15 min on ice and then centrifuged at 21 100 g at 4°C for 1 h. The extracts were subjected to immunoblot using antibodies for MKRN2 (1:2000 dilution; catalog# NB100-55250, Novus Biologicals), PERP (1:500 dilution; catalog# AB_48032, Abcam), p53 (DO-7) (1:1000 dilution; catalog# 48818, CST), and anti-p53 (acetyl-K120) (1:1000 dilution; catalog# HW-186, SAB, Inc., Nanjing, China) as described above.

Techniques: Isolation, Gene Expression, Quantitative Proteomics, Knock-Out

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: DEGs and PERP expression levels in Mkrn 2-KO cells and tissues. ( a ) Expression levels of DEGs for the Mkrn 2-KO and Mkrn 2-WT MEFs are analyzed by qRT-PCR. Data are presented and analyzed by the Student's t -test from three independent experiments performed in triplicate. ** Significant difference compared with WT group ( P < 0.01). 293T cells were co-transfected with an Mkrn2 shRNA (shMkrn2) or negative control (Mock) vector plus an MKRN2 expression vector or control vector. ( b ) qRT-PCR is used to analyze the mRNA expression of PERP . Data are presented as mean ± s.d. from three independent experiments performed in triplicate. ( c ) Western blot is used to analyze the protein expression of PERP, densitometric analysis of Western blot signals (right). ( b and c ) Results are presented and analyzed by the two-way ANOVA, *Significant difference at P < 0.05, **Significant difference at P < 0.01. ( d ) Correlation between MKRN 2 and PERP expression levels in GEO dataset GSE6872 (contains sperm samples), as analyzed by Pearson's correlation analysis. DEG: differential expression gene; Mkrn 2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to peripheral myelin protein 22 (PMP22); qRT-PCR: quantitative real-time polymerase chain reaction; s.d.: standard deviation; MEF: mouse embryonic fibroblast; GEO: Gene Expression Omnibus.

Article Snippet: The tissues lysates were subjected to three sets of 15-s pulses between pulses for 15 min on ice and then centrifuged at 21 100 g at 4°C for 1 h. The extracts were subjected to immunoblot using antibodies for MKRN2 (1:2000 dilution; catalog# NB100-55250, Novus Biologicals), PERP (1:500 dilution; catalog# AB_48032, Abcam), p53 (DO-7) (1:1000 dilution; catalog# 48818, CST), and anti-p53 (acetyl-K120) (1:1000 dilution; catalog# HW-186, SAB, Inc., Nanjing, China) as described above.

Techniques: Expressing, Quantitative RT-PCR, Transfection, shRNA, Negative Control, Plasmid Preparation, Control, Western Blot, Quantitative Proteomics, Knock-Out, Real-time Polymerase Chain Reaction, Standard Deviation, Gene Expression

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: Correlation of PERP levels with spermatogenesis. ( a ) Relative expression levels of the Mkrn 2 in normal and OAT sperm samples from the GSE6872 dataset. Data are presented and analyzed by two-tailed, unpaired Student's t -test. ** Significant differences compared with WT group ( P < 0.01). ( b ) Enrichment and ranking of genes in the PERP -high and PERP -low expression groups of the GSE6872 dataset are assessed by GSEA analysis (C5: GO gene sets; BP: GO biological process; gene set: male gamete generation) and the corresponding clustering heatmap of genes that contribute most to enrichment score is on the right. ( c ) Enrichment and ranking of genes in the PERP -high and PERP -low expression groups of the GSE6872 dataset are assessed by GSEA analysis (H: hallmark gene set; gene set: spermatogenesis) and the corresponding clustering heatmap of genes that contribute most to enrichment score is on the right ( PERP- low datasets: GSM158476, GSE158477, and GSM158479 and PERP -high datasets: GSM158478, GSM158480, GSM158481, GSM158482, and GSM158483). ( d ) Correlations between spermatogenic signature scores and PERP expression levels are determined by Spearman's rank correlation analysis. Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; GSEA: gene set enrichment analysis; OAT: oligoasthenoteratozoospermia.

Article Snippet: The tissues lysates were subjected to three sets of 15-s pulses between pulses for 15 min on ice and then centrifuged at 21 100 g at 4°C for 1 h. The extracts were subjected to immunoblot using antibodies for MKRN2 (1:2000 dilution; catalog# NB100-55250, Novus Biologicals), PERP (1:500 dilution; catalog# AB_48032, Abcam), p53 (DO-7) (1:1000 dilution; catalog# 48818, CST), and anti-p53 (acetyl-K120) (1:1000 dilution; catalog# HW-186, SAB, Inc., Nanjing, China) as described above.

Techniques: Expressing, Two Tailed Test, Knock-Out

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: Association of PERP and p53 expression levels in Mkrn 2-KO cells and tissues. ( a ) Expression levels of p53 are analyzed by GEO dataset GSE6872. Data are presented and analyzed by two-tailed, unpaired Student's t -test. * Significant difference compared with the normal group ( P < 0.05). ( b ) Expression levels of MKRN2, p53, and PERP in MEFs are analyzed by Western blot (left), densitometric analysis of Western blot signals (right). ( c ) Expression levels of p53 and PERP in MEFs transfected with a p53 vector or CON as indicated are analyzed by Western blot (left), densitometric analysis of Western blot signals (right). ( d ) PERP expression levels are determined by Western blot in 293T cells co-transfected with vector and control, Mkrn2 and CON, Mkrn2 and p53, respectively (left), densitometric analysis of Western blot signals (right). ( c and d ) Results are presented and analyzed by the two-way ANOVA from three independent experiments and each performed for three times. * Significant difference at P < 0.05, ** Significant difference at P < 0.01. CON: negative control vector; Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; GEO: Gene Expression Omnibus; MEF: mouse embryonic fibroblast.

Article Snippet: The tissues lysates were subjected to three sets of 15-s pulses between pulses for 15 min on ice and then centrifuged at 21 100 g at 4°C for 1 h. The extracts were subjected to immunoblot using antibodies for MKRN2 (1:2000 dilution; catalog# NB100-55250, Novus Biologicals), PERP (1:500 dilution; catalog# AB_48032, Abcam), p53 (DO-7) (1:1000 dilution; catalog# 48818, CST), and anti-p53 (acetyl-K120) (1:1000 dilution; catalog# HW-186, SAB, Inc., Nanjing, China) as described above.

Techniques: Expressing, Two Tailed Test, Western Blot, Transfection, Plasmid Preparation, Control, Negative Control, Knock-Out, Gene Expression

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: p53/PERP expression levels in Mkrn 2-KO mouse testes. ( a ) TUNEL assay to assess the levels of apoptosis in Mkrn 2 KO versus WT mouse testis. Immunohistochemical staining of ( b ) p53 and ( c ) PERP expression in the testes of Mkrn 2-KO and Mkrn 2-WT mice. Scale bars=50 μm. ( d ) Western blot assay to test expression levels of MKRN2, p53, and PERP (left); and the different densitometric levels between two groups: Mkrn 2-KO and Mkrn 2-WT mouse testes are analyzed by the two-tailed, unpaired Student's t -test to compare the data of the two groups from three independent experiments (right). ** Significant difference at P < 0.01. Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; MEF: mouse embryonic fibroblast; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: The tissues lysates were subjected to three sets of 15-s pulses between pulses for 15 min on ice and then centrifuged at 21 100 g at 4°C for 1 h. The extracts were subjected to immunoblot using antibodies for MKRN2 (1:2000 dilution; catalog# NB100-55250, Novus Biologicals), PERP (1:500 dilution; catalog# AB_48032, Abcam), p53 (DO-7) (1:1000 dilution; catalog# 48818, CST), and anti-p53 (acetyl-K120) (1:1000 dilution; catalog# HW-186, SAB, Inc., Nanjing, China) as described above.

Techniques: Expressing, TUNEL Assay, Immunohistochemical staining, Staining, Western Blot, Two Tailed Test, Knock-Out