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. 2011 Jul 8;43(1):19–32. doi: 10.1016/j.molcel.2011.04.029

Figure 2.

Figure 2

NO Reduced Phosphorylation of JNK1 and Bcl-2, Leading to Increased Bcl-2–Beclin 1 and Decreased Beclin 1–hVps34 Interactions

(A) NO donors reduced JNK1 phosphorylation in HeLa cells, as analyzed by immunoprecipitation with anti-JNK1 agarose-conjugated beads and immunoblotting with anti-phospho-JNK antibody.

(B) NO donors reduced Bcl-2 phosphorylation in HeLa cells, as analyzed by immunoblotting with anti-phospho-Bcl-2 antibody.

(C) Immunoblot analysis with anti-phospho-Bcl-2 antibody shows that DETA NONOate could not reduce phospho-Bcl-2 in Flag-CA JNK1-transfected HeLa cells, where phospho-Bcl-2 was higher compared to mock-transfected cells.

(D and E) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Myc antibody shows that NO donors increased the interaction of Flag-Beclin 1 with WT Myc-Bcl-2 (D), but not with AAA Myc-Bcl-2 (E), in HeLa cells transfected with WT Myc-Bcl-2 (D) or AAA Myc-Bcl-2 (E) along with either empty Flag or Flag-Beclin 1. Asterisk denotes IgG band (E).

(F) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that NO donors decreased Flag-Beclin 1–hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1.

(G) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that AAA Myc-Bcl-2 decreased Flag-Beclin 1–hVps34 interaction in the presence (right panel) or absence (left panel) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1, and with pcDNA3.1 or AAA Myc-Bcl-2. Graphical data denote mean ± SEM.