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. 2004 May;16(5):1235–1250. doi: 10.1105/tpc.020719

Figure 3.

Figure 3.

siRNA Coimmunoprecipitate with P19 in Arabidopsis and in Mammalian Cells.

(A) Immunodetection of P19:HA and P19m:HA (anti-HA antibody) in seedlings of CHS-RNAi plants that had been transformed either with the P19:HA or with the P19m:HA construct. The proteins accumulate to similar levels in the two lines shown here. Coomassie blue staining of the immunoblot indicates equal protein loading.

(B) Anthocyanin accumulation and RNA gel blot analysis of the low molecular weight RNA fraction extracted from the P19:HA and P19m:HA seedlings. Hybridization was with a CHS cDNA probe. nt, nucleotides.

(C) Total proteins were extracted from seedlings. P19:HA and P19m:HA were immunoprecipitated with an anti-HA antibody, and the presence of each protein was assayed by protein gel blot analysis of the immunoprecipitated (IP) fractions (top panel). After deproteinization, nucleic acids extracted from the IP fractions were subjected to RNA gel blot analysis using a CHS cDNA probe (bottom panel).

(D) Dual-LUC assay performed in human Hela cells transfected with pGL3-CMV (encoding the firefly LUC) and pRL-CMV (encoding the renilla LUC) together with pSG5m (mock) or with pSGP19 (P19), psGP19HA (P19:HA), or psGP19mHA (P19m:HA). Twenty-four hours later, cells were supertransfected with 0 (−) or 300 ng (+) of siRNAs directed against the firefly LUC mRNA. The renilla LUC mRNA is not targeted by these siRNAs and is therefore used as a reference in this assay. For each treatment, the luminescence ratio firefly/renilla was calculated. This ratio was then normalized to the luminescence values from a transfection experiment performed in parallel, in which anti-LUC siRNAs were omitted. This provided a relative LUC activity. For each treatment, results of two independent assays are presented. Values from each assay were from duplicate independent transfections.

(E) Human Hela cells were transfected with pSGP19:HA together with anti-LUC siRNAs. Two days later, P19:HA was immunoprecipitated with an anti-HA antibody, and the presence of the protein was assayed by protein gel blot analysis (top panel). After deproteinization, nucleic acids were extracted from the immunoprecipitated fractions (IP) and subjected to RNA gel blot analysis using radiolabeled anti-LUC siRNAs as probe (bottom panel). nt, nucleotides.

(F) Same experimental set up as in (D) but performed with pSG5m expression vectors for the P25, P38, and P15 proteins. The values in each bar are from three independent experiments conducted in triplicate.