Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R disruption exacerbates synucleinopathies in the nigrostriatal pathway in an αSyn AAV mouse model. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN and sacrificed 8 weeks later: A Immunoblot of human αSyn species in Triton X-100-soluble and -insoluble SDS-soluble fractions in ipsilateral ventral midbrain and striatum and B quantification of αSyn monomers and oligomers. n = 3 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. C p-αSyn staining and D quantification of p-αSyn aggregates in the ipsilateral SN. n = 3 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. Two-tail Student’s t -test. Scale bar, 50 µm. MC1R e/e and WT mice were injected unilaterally with BiFC human WT αSyn AAV into the SN and sacrificed 8 weeks later: E VenusYFP fluorescence before and after proteinase K treatment and F quantification of venusYFP fluorescence density in the ipsilateral SN. n = 4 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. Scale bar, 50 µm. G Thioflavin-S staining and H quantification of thioflavin-S fluorescence density in the ipsilateral SN. n = 4 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. Two-tail Student’s t -test. Scale bar, 50 µm. * P < 0.05, ** P < 0.01

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Injection, Western Blot, Staining, Fluorescence

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R disruption amplifies microglia activation and alters Nrf2 response to αSyn overexpression in the nigrostriatal pathway. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN and sacrificed 8 weeks later: A Iba1 staining and B morphological classification and quantification of iba1-positive cells in the SN. n = 4 mice/group. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 30 µm. C IL-1a, IL-6, TNFα, and ICAM1 mRNA levels in ventral midbrain. Measurements were normalized by dividing values by the mean of the WT contralateral side. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. D Representative oxyblots for protein carbonyls and the corresponding Ponceau S staining in the ipsilateral ventral midbrain and E quantification of band density. Measurements were normalized by dividing values by the mean of WT control and multiplying by 100. Two-tail Student’s t -test. n = 3 mice/group. F Immunoblot for Nrf2 using ventral midbrain tissue and G quantification of Nrf2 band density in original values or H normalized to contralateral side by dividing values by the mean of the contralateral side and multiplying by 100. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. I SN sections double-stained for Nrf2 and TH and J quantification of nuclear and cytoplasmic Nrf2. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. Scale bar, 20 µm. K mRNA levels of Nrf2 target genes HO-1, NQO1, GCLM, and GCLC in the ipsilateral ventral midbrain. Measurements were normalized by dividing values by the mean of WT control. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Activation Assay, Over Expression, Injection, Staining, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: αSyn-induced dopaminergic neurotoxicity is exacerbated by MC1R disruption and reversed by human MC1R transgene. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN. A Contralateral and ipsilateral turns induced by amphetamine 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. B Striatal dopamine content 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. C TH staining and D stereological quantification of TH-positive and -negative cells in the SN 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. Scale bar, 100 µm. MC1R e/e Tg and MC1R e/e mice were injected unilaterally with human WT αSyn AAV into the SN. E Striatal dopamine content 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–7 mice/group. F TH staining and G stereological quantification of TH-positive and negative cells in the SN 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–7 mice/group. Scale bar, 100 µm. H p-αSyn staining and I quantification of p-αSyn aggregates in the ipsilateral SNpc 12 weeks post-AAV injection. Measurements were normalized by dividing values by the mean of the MC1R e/e mice and multiplying by 100. Two-tail Student’s t test. n = 4 mice/group. Scale bar, 50 µm. J Iba1 staining and morphological classification and K quantification of iba1-positive cells in the SNpc 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. Scale bar, 30 µm. L Nrf2 and TH double-labeling and (M) quantification of nuclear and cytoplasmic Nrf2 in the SNpc 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. Scale bar, 20 µm. N mRNA levels of Nrf2 target genes HO-1, NQO1, GCLC, and GCLM in the ipsilateral ventral midbrain 12 weeks post-AAV injection. Measurements were normalized by dividing values by the mean of the MC1R e/e mice. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Injection, Staining, Labeling

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R agonists protect against αSyn-induced dopaminergic neurotoxicity. C57Bl/6 J mice were injected with αSyn or empty vector (Vec) AAV, treated subcutaneously with 20 mg/kg BMS-470539 (BMS-) or saline for 4 weeks, and sacrificed 16 weeks post-AAV injection. A Striatal dopamine content. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–8 mice/group. B Stereological quantification of TH-positive cells in the SN. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–8 mice/group. C p-αSyn staining and D quantification of p-αSyn aggregates in the ipsilateral SN. Measurements were normalized by dividing values by the mean of the vehicle-treated group and multiplying by 100. Two-tailed Student’s t test. n = 4 mice/group. Scale bar, 50 µm. WT and MC1R e/e mice were injected unilaterally with 3 nmol NDP-MSH into the striatum and then with αSyn AAV into the SN. Mice were sacrificed 16 weeks post-AAV injection. E Contralateral and ipsilateral turns induced by amphetamine 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. F Striatal dopamine content. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. G Stereological quantification of TH-positive cells in the SN. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. H p-αSyn staining and I quantification of p-αSyn aggregates in the ipsilateral SN. Measurements were normalized by dividing values by the mean of the vehicle-treated group and multiplying by 100. Two-tail Student’s t test. n = 4 mice/group. Scale bar, 50 µm. J Morphological classification and quantification of iba1-positive cells in the SN in WT mice. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. K Nuclear and cytoplasmic Nrf2 ratio in the SN in WT mice. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Injection, Plasmid Preparation, Staining, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R activation alleviates αSyn oligomerization and neurotoxicity by activating Nrf2 in vitro. HEK293T cells were co-transfected with αSyn and MC1R or vector (GPRC5A-Tango). Non-transfected cells served as controls (NC): A Immunoblot and quantification of MC1R and Nrf2. One-way ANOVA followed by Tukey's post hoc test. B Nrf2 mRNA levels. One-way ANOVA followed by Tukey's post hoc test. C Immunoblot and quantification of pCREB and CBP. One-way ANOVA followed by Tukey's post hoc test. D ChIP-qPCR analysis of pCREB binding in the Nrf2 promoter. Chromatins were immunoprecipitated using pCREB or IgG as negative control (Neg). Values were calculated by subtracting the cycle threshold (Ct) values for immunoprecipitated DNA from adjusted Ct of input DNA to get delta Ct, followed by raising 2 to the power of the delta Ct and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. E Nrf2 staining and quantification of nuclear and cytoplasmic Nrf2. Nuclei were stained with DAPI. One-way ANOVA followed by Tukey's post hoc test. Scale bar, 10 µm. F Nrf2 target gene HO-1 mRNA levels. One-way ANOVA followed by Tukey's post hoc test. G and H Immunoblot and quantification of αSyn species. Measurements were normalized by dividing values by the mean of vector control and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. HEK293T cells were transfected with αSyn, MC1R or vector control, and shNrf2 RNA or scRNA control: G Immunoblot and I and J quantification of MC1R and Nrf2. One-way ANOVA followed by Tukey's post hoc test. G Immunoblot and K quantification of αSyn species. Measurements were normalized by dividing values by the mean of vector control and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. Primary cortical neurons were prepared from embryonic day 16–17 WT mice: L MAP2 and MC1R staining at DIV5. Scale bar, 10 µm. (M) Nrf2 mRNA levels in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-tail Student’s t test. N LDH release in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA and treated with PBS or NDP-MSH. Two-way ANOVA followed by Tukey’s post hoc test. O MAP2 staining and N quantification of MAP2-postive cells in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 10 µm. Visual field area = 0.13508 mm 2 . * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not statistically significant. n = 3 replicates. Experiments were repeated ≥ 3 times

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Activation Assay, In Vitro, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Immunoprecipitation, Negative Control, Staining, Transduction

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R is present in dopaminergic neurons in humans and is reduced in patients with PD. A Immunohistochemistry for MC1R and TH in the SN of a 60-year-old control individual, PMI 15 h. Control sections were processed in the same manner, except primary or secondary antibodies were omitted. Scale bars, top 100 µm, bottom 25 µm. B Fluorescence double-staining for MC1R and TH or GFAP in the SN of a 63-year-old control individual, PMI 16 h. Scale bar, 50 µm. C Immunohistochemistry for MC1R and TH in the SN of a 91-year-old control individual, PMI 8 h, and an 84-year-old PD patient, PMI 6 h. Scale bar, 50 µm. D Fluorescence double-staining of MC1R and TH in the SN of an 87-year-old control individual, PMI 48 h, and a 91-year-old PD patient, PMI 32 h. Scale bar, 50 µm. E Immunoblot for MC1R and TH using SN tissue from control individuals and PD patients and F quantification of band density, n = 4/group; G Immunoblot for Nrf2 using SN tissue from control individuals and PD patients and H quantification of band density, n = 3/group. Actin as a loading control. Two-tail Student’s t test. Age (years)/PMI (h): 63/16, 87/48, 91/8, and 60/15 for control 1–4; 62/33, 91/32, 84/6, and 69/17 for PD 1–4. * P < 0.05

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Fluorescence, Double Staining, Western Blot

Journal: BMC Medicine

Article Title: Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth

doi: 10.1186/s12916-022-02391-4

Figure Lengend Snippet: Increased STAT5B expression and phosphorylation during syncytialization of human trophoblasts. A The putative STAT5B binding sites at the ADAM12 promoter revealed by in silico analysis. TSS, transcription start site. B – E Immunohistochemical staining showed that total STAT5B and phosphorylated STAT5B were mainly localized in the syncytiotrophoblast (STB) but not the cytotrophoblast (CTB) of human placental villi at both early and term pregnancies. Total STAT5B was localized in both cytoplasm and nucleus, while phosphorylated STAT5B was localized mainly in the nucleus of syncytiotrophoblast. Non-immune serum served as negative control (NC). Scale bars, 20 μm. F Increased STAT5B mRNA ( n = 6) and protein ( n = 7) abundance during syncytialization of cultured human trophoblasts. (G) Increased STAT5B phosphorylation during syncytialization of cultured human trophoblasts ( n = 4). Top panels of F and G are representative immunoblots. Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs 3 hrs

Article Snippet: After blocking with 5% nonfat milk, the membrane was probed with primary antibodies against EZH2 (1:2000, Cell Signaling, #5246), STAT5B (1:250, Invitrogen, #712500), phosphorylated STAT5B at Tyr694 (1:1000, Cell Signaling, #9351), ADAM12-S (1:1000, Abcam, #223476) and GAPDH (1:10,000, Proteintech, #60004–1) respectively overnight at 4 °C.

Techniques: Expressing, Binding Assay, In Silico, Immunohistochemical staining, Staining, Negative Control, Cell Culture, Western Blot

Journal: BMC Medicine

Article Title: Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth

doi: 10.1186/s12916-022-02391-4

Figure Lengend Snippet: Role of STAT5B in the regulation of ADAM12-S expression during syncytialization of human trophoblasts. A – C siRNA-mediated knockdown of STAT5B expression increased the abundance of ADAM12S mRNA ( n = 4) and protein in cultured trophoblasts ( n = 6) as well as secreted ADAM12-S in trophoblast culture medium ( n = 3). Randomly scrambled siRNA served as negative control (NC). Top panels of A – C are representative immunoblots. D Increased enrichment of STAT5B at ADAM12 promoter upon syncytialization of cultured trophoblast ( n = 4). Top panel of D illustrates aligning positions of primers used in ChIP assay. TSS, transcription start site. Data are means ± SEM. * P < 0.05, *** P < 0.001 vs NC or 3 hrs

Article Snippet: After blocking with 5% nonfat milk, the membrane was probed with primary antibodies against EZH2 (1:2000, Cell Signaling, #5246), STAT5B (1:250, Invitrogen, #712500), phosphorylated STAT5B at Tyr694 (1:1000, Cell Signaling, #9351), ADAM12-S (1:1000, Abcam, #223476) and GAPDH (1:10,000, Proteintech, #60004–1) respectively overnight at 4 °C.

Techniques: Expressing, Cell Culture, Negative Control, Western Blot

Journal: BMC Medicine

Article Title: Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth

doi: 10.1186/s12916-022-02391-4

Figure Lengend Snippet: Role of EGF/STAT5B pathway in the induction of ADAM12-S expression during syncytialization of human trophoblast. A Increased EGFR mRNA and protein abundance during syncytialization ( n = 4). B Time course of EGF (10 ng/mL) on STAT5B phosphorylation in syncytiotrophoblasts ( n = 4). C Effects of EGF (10 ng/mL, 24 hrs) on STAT5B mRNA ( n = 3) and protein abundance in syncytiotrophoblasts ( n = 4). D siRNA-mediated knockdown of STAT5B attenuated basal and EGF (10 ng/mL)-induced ADAM12S mRNA expression ( n = 5) and ADAM12-S secretion ( n = 5) in cultured trophoblasts. Randomly scrambled siRNA served as negative control (NC). Top panel of each figure is the representative immunoblot. Data are means ± SEM. * P < 0.05, ** P < 0.01 vs corresponding control; ## P < 0.01 vs EGF-treated group

Article Snippet: After blocking with 5% nonfat milk, the membrane was probed with primary antibodies against EZH2 (1:2000, Cell Signaling, #5246), STAT5B (1:250, Invitrogen, #712500), phosphorylated STAT5B at Tyr694 (1:1000, Cell Signaling, #9351), ADAM12-S (1:1000, Abcam, #223476) and GAPDH (1:10,000, Proteintech, #60004–1) respectively overnight at 4 °C.

Techniques: Expressing, Cell Culture, Negative Control, Western Blot

Journal: BMC Medicine

Article Title: Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth

doi: 10.1186/s12916-022-02391-4

Figure Lengend Snippet: Increased STAT5B expression and phosphorylation during syncytialization of human trophoblasts. A The putative STAT5B binding sites at the ADAM12 promoter revealed by in silico analysis. TSS, transcription start site. B – E Immunohistochemical staining showed that total STAT5B and phosphorylated STAT5B were mainly localized in the syncytiotrophoblast (STB) but not the cytotrophoblast (CTB) of human placental villi at both early and term pregnancies. Total STAT5B was localized in both cytoplasm and nucleus, while phosphorylated STAT5B was localized mainly in the nucleus of syncytiotrophoblast. Non-immune serum served as negative control (NC). Scale bars, 20 μm. F Increased STAT5B mRNA ( n = 6) and protein ( n = 7) abundance during syncytialization of cultured human trophoblasts. (G) Increased STAT5B phosphorylation during syncytialization of cultured human trophoblasts ( n = 4). Top panels of F and G are representative immunoblots. Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs 3 hrs

Article Snippet: After blocking with 5% nonfat milk, the membrane was probed with primary antibodies against EZH2 (1:2000, Cell Signaling, #5246), STAT5B (1:250, Invitrogen, #712500), phosphorylated STAT5B at Tyr694 (1:1000, Cell Signaling, #9351), ADAM12-S (1:1000, Abcam, #223476) and GAPDH (1:10,000, Proteintech, #60004–1) respectively overnight at 4 °C.

Techniques: Expressing, Binding Assay, In Silico, Immunohistochemical staining, Staining, Negative Control, Cell Culture, Western Blot

Journal: BMC Medicine

Article Title: Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth

doi: 10.1186/s12916-022-02391-4

Figure Lengend Snippet: Role of STAT5B in the regulation of ADAM12-S expression during syncytialization of human trophoblasts. A – C siRNA-mediated knockdown of STAT5B expression increased the abundance of ADAM12S mRNA ( n = 4) and protein in cultured trophoblasts ( n = 6) as well as secreted ADAM12-S in trophoblast culture medium ( n = 3). Randomly scrambled siRNA served as negative control (NC). Top panels of A – C are representative immunoblots. D Increased enrichment of STAT5B at ADAM12 promoter upon syncytialization of cultured trophoblast ( n = 4). Top panel of D illustrates aligning positions of primers used in ChIP assay. TSS, transcription start site. Data are means ± SEM. * P < 0.05, *** P < 0.001 vs NC or 3 hrs

Article Snippet: After blocking with 5% nonfat milk, the membrane was probed with primary antibodies against EZH2 (1:2000, Cell Signaling, #5246), STAT5B (1:250, Invitrogen, #712500), phosphorylated STAT5B at Tyr694 (1:1000, Cell Signaling, #9351), ADAM12-S (1:1000, Abcam, #223476) and GAPDH (1:10,000, Proteintech, #60004–1) respectively overnight at 4 °C.

Techniques: Expressing, Cell Culture, Negative Control, Western Blot

Journal: BMC Medicine

Article Title: Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth

doi: 10.1186/s12916-022-02391-4

Figure Lengend Snippet: Role of EGF/STAT5B pathway in the induction of ADAM12-S expression during syncytialization of human trophoblast. A Increased EGFR mRNA and protein abundance during syncytialization ( n = 4). B Time course of EGF (10 ng/mL) on STAT5B phosphorylation in syncytiotrophoblasts ( n = 4). C Effects of EGF (10 ng/mL, 24 hrs) on STAT5B mRNA ( n = 3) and protein abundance in syncytiotrophoblasts ( n = 4). D siRNA-mediated knockdown of STAT5B attenuated basal and EGF (10 ng/mL)-induced ADAM12S mRNA expression ( n = 5) and ADAM12-S secretion ( n = 5) in cultured trophoblasts. Randomly scrambled siRNA served as negative control (NC). Top panel of each figure is the representative immunoblot. Data are means ± SEM. * P < 0.05, ** P < 0.01 vs corresponding control; ## P < 0.01 vs EGF-treated group

Article Snippet: After blocking with 5% nonfat milk, the membrane was probed with primary antibodies against EZH2 (1:2000, Cell Signaling, #5246), STAT5B (1:250, Invitrogen, #712500), phosphorylated STAT5B at Tyr694 (1:1000, Cell Signaling, #9351), ADAM12-S (1:1000, Abcam, #223476) and GAPDH (1:10,000, Proteintech, #60004–1) respectively overnight at 4 °C.

Techniques: Expressing, Cell Culture, Negative Control, Western Blot