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. 2007 Jan 8;27(6):2324–2342. doi: 10.1128/MCB.02300-06

FIG. 3.

FIG. 3.

Caprin-1 interacts with G3BP-1 through an evolutionarily conserved peptide motif. (A) G3BP1 binds to a conserved peptide motif in Caprin-1. 293T cells were cotransfected with plasmids expressing Flag-G3BP-1 and various fragments of GFP-Caprin-1 comprising amino acids 1 to 327, 1 to 606, 352 to 606, or 352 to 380, as indicated. Cell lysates were subjected to anti-Flag IP, and precipitated proteins were eluted from the beads and subjected to SDS-PAGE and immunoblotting with anti-GFP, anti-Flag, or anti-G3BP-1 antibodies. (B) (i) Conserved features of Caprin-1- and insect HR-1-containing proteins. The amino-terminal MPSA motifs, the central conserved motif, and the RGG motifs are highlighted. (ii) Also shown is an alignment of the central conserved G3BP-1 binding motif in three insect HR-1-containing proteins and vertebrate Caprin-1 and Caprin-2 together with the core consensus. (C) Peptides containing the G3BP-1-binding Caprin-1 motif compete with Caprin-1 for binding to G3BP-1. (i) Sequence of the core consensus peptide and an extended consensus peptide from the G3BP-1 binding motif from human Caprin-1. (ii) 293T cells were transfected with plasmids expressing Flag-G3BP-1 and GFP-Caprin-1 and lysates were subjected to anti-Flag IP. Washed beads, with bound GFP-Caprin-1 and Flag-G3BP-1 were agitated in 1 ml buffer containing the indicated peptides at 50 μM or 200 μM or buffer alone for 90 min at 4°C. The proteins on the beads were eluted and immunoblotted with anti-GFP and anti-Flag antibodies. (iii) 293T cells were transfected with plasmids expressing Flag-G3BP-1 and GFP-Caprin-1, and lysates were subjected to anti-Flag IP. Washed beads, with bound GFP-Caprin-1 and Flag-G3BP-1 were agitated in 1 ml buffer containing the indicated peptide at 380 μM or buffer alone for 90 min at 4°C. Also shown is a coprecipitation of GFP-Caprin and Flag-G3BP-1, incubated with 100 μg of RNase A in 1 ml of buffer for 60 min. The proteins on the beads were eluted and subjected to SDS-PAGE and immunoblotting with anti-GFP and anti-Flag antibodies.