Journal: PLoS Biology

Article Title: Angiogenic Factor AGGF1 Activates Autophagy with an Essential Role in Therapeutic Angiogenesis for Heart Disease

doi: 10.1371/journal.pbio.1002529

Figure Lengend Snippet: (A) Western blot analysis for phosphorylation of JNK with protein extracts from HUVECs treated with different concentrations of AGGF1 for 4 h. (B) Activation of JNK in HUVECs treated with AGGF1 protein (500 ng/ ml) for different time points. (C) JNK inhibitors II and III inhibited AGGF1-induced autophagy (LC3-II levels) and activation of JNK. (D) Co-immunoprecipitation to assess the assembly of Beclin1-Vps34-Atg14L complex. Immunoprecipitation, beclin 1 antibody, or control IgG; Western blot, individual antibodies for Vsp34, Atg14L, or beclin 1. (E) Vps34 lipid kinase activity with ELISA assays ( n = 3/group). (F) Autophagic activity assay for membrane-associated PI(3)K (GFP-2xFYVE dots). HUVECs were transfected with GFP-2xFYVE for 24 h and then treated with JNK inhibitor II or control DMSO, followed by treatment with AGGF1 or IgG ( n = 6/group). Scale bar = 10 μm. (G) Autophagic activity assay for degradation of long-lived, damaged proteins by 3 H-leucine release in HUVECs ( n = 6/group). ( H ) JNK1 siRNA inhibited AGGF1-induced autophagy (LC3-II levels) and expression of JNK1. ( I ) Co-immunoprecipitation to assess the assembly of the Beclin1-Vps34-Atg14L complex by JNK1 siRNA. ( J ) Atg14L siRNA inhibited AGGF1-induced autophagy (LC3-II levels) and expression of Atg14L. ( K ) Co-immunoprecipitation to assess the assembly of the Beclin1-Vps34-Atg14L complex by Atg14L siRNA. Underlying data are shown in .

Article Snippet: Exogenous Vps34 expressed in HUVECs was immunoprecipitated using an anti-HA antibody, measured using the class III PI3K assay, and was carried out using the Class III PI3-kinase ELISA kit (Echelon, UT, US).

Techniques: Western Blot, Activation Assay, Immunoprecipitation, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing