Journal: Acta Biochimica et Biophysica Sinica

Article Title: ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury

doi: 10.3724/abbs.2024120

Figure Lengend Snippet: Table 1 Primary and secondary antibodies used in this study

Article Snippet: Endogenous immunoprecipitation was performed by incubating cell lysates with anti-ZIPK antibody (1:500; 30656-1-AP; Proteintech) or anti-STAT5A antibody (1:500; 366459-1-Ig; Proteintech), using anti-IgG antibody (1:500; 30000-0-AP; Proteintech) as the negative control, supplemented with 50 μL of protein A/G Dynabeads (Thermo Fisher Scientific) at 4°C overnight.

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury

doi: 10.3724/abbs.2024120

Figure Lengend Snippet: Table 2 Sequence of primers used for real-time RT-PCR

Article Snippet: Endogenous immunoprecipitation was performed by incubating cell lysates with anti-ZIPK antibody (1:500; 30656-1-AP; Proteintech) or anti-STAT5A antibody (1:500; 366459-1-Ig; Proteintech), using anti-IgG antibody (1:500; 30000-0-AP; Proteintech) as the negative control, supplemented with 50 μL of protein A/G Dynabeads (Thermo Fisher Scientific) at 4°C overnight.

Techniques: Sequencing, Quantitative RT-PCR

Journal: Acta Biochimica et Biophysica Sinica

Article Title: ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury

doi: 10.3724/abbs.2024120

Figure Lengend Snippet: ZIPK is involved in the expressions of P53 and ROS accumulation in HUVECs induced by high glucose (A) Western blot analysis of STAT5A, P53, and NOS2 protein levels following high glucose treatment and reduced STAT5A expression. (B) Statistical analysis of protein expression levels (n = 3 per group). (C) qPCR detection of the mRNA expression levels of STAT5A, P53, and NOS2 under the indicated conditions (n = 5 per group). (D) Flow cytometry detection of intracellular ROS levels over 24 h in the experimental groups. (E) Statistical analysis of ROS levels (n = 3 per group). **P < 0.01 vs the control group, ## P < 0.01 vs the HG group. HG, high glucose.

Article Snippet: Endogenous immunoprecipitation was performed by incubating cell lysates with anti-ZIPK antibody (1:500; 30656-1-AP; Proteintech) or anti-STAT5A antibody (1:500; 366459-1-Ig; Proteintech), using anti-IgG antibody (1:500; 30000-0-AP; Proteintech) as the negative control, supplemented with 50 μL of protein A/G Dynabeads (Thermo Fisher Scientific) at 4°C overnight.

Techniques: Western Blot, Expressing, Flow Cytometry, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury

doi: 10.3724/abbs.2024120

Figure Lengend Snippet: ZIPK is involved in the increase in P53 expression and ROS levels in HUVECs induced by high glucose through STAT5A (A) Western blot analysis of ZIPK, STAT5A, P53, and NOS2 protein levels following ZIPK overexpression and reduced STAT5A expression. (B) Statistical analysis of protein expression levels (n = 3 per group). (C) qPCR detection of the mRNA expression levels of STAT5A, P53, and NOS2 under the indicated conditions (n = 5 per group). (D) Flow cytometry detection of intracellular ROS levels over 24 h in the experimental groups. (E) Statistical analysis of ROS levels (n = 3 per group). **P < 0.01 vs the control group, ## P < 0.01 vs the ZIPK-OE group.

Article Snippet: Endogenous immunoprecipitation was performed by incubating cell lysates with anti-ZIPK antibody (1:500; 30656-1-AP; Proteintech) or anti-STAT5A antibody (1:500; 366459-1-Ig; Proteintech), using anti-IgG antibody (1:500; 30000-0-AP; Proteintech) as the negative control, supplemented with 50 μL of protein A/G Dynabeads (Thermo Fisher Scientific) at 4°C overnight.

Techniques: Expressing, Western Blot, Over Expression, Flow Cytometry, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury

doi: 10.3724/abbs.2024120

Figure Lengend Snippet: ZIPK interacts with STAT5A (A) Simulated diagram using AlphaFold3 showing the binding of ZIPK to the single-stranded DNA of the STAT5A promoter region. (B) Analysis of AlphaFold3 showing the binding of ZIPK to the single-stranded DNA of the STAT5A promoter region, with an interface-predicted template model (ipTM) = 0.19 and a predicted template model (pTM) = 0.28. (C) Simulated diagram using AlphaFold3 illustrating the binding of ZIPK to the double-stranded DNA of the STAT5A promoter region. (D) Analysis of AlphaFold3 demonstrating the binding of ZIPK to the double-stranded DNA of the STAT5A promoter region, with ipTM = 0.27 and pTM = 0.31. (E) Simulated diagram using AlphaFold3 showing the interaction between ZIPK and STAT5A. (F) Analysis of AlphaFold3 revealed an interaction between ZIPK and STAT5A, with ipTM = 0.16 and pTM = 0.49. (G) The interaction between ZIPK and STAT5A in HUVECs was determined by immunoprecipitation. (H) Confocal microscopy confirmed the colocalization of ZIPK and STAT5A in HUVECs.

Article Snippet: Endogenous immunoprecipitation was performed by incubating cell lysates with anti-ZIPK antibody (1:500; 30656-1-AP; Proteintech) or anti-STAT5A antibody (1:500; 366459-1-Ig; Proteintech), using anti-IgG antibody (1:500; 30000-0-AP; Proteintech) as the negative control, supplemented with 50 μL of protein A/G Dynabeads (Thermo Fisher Scientific) at 4°C overnight.

Techniques: Binding Assay, Immunoprecipitation, Confocal Microscopy