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. 2010 May 27;285(30):23177–23185. doi: 10.1074/jbc.M110.109868

FIGURE 5.

FIGURE 5.

HMG2L1 interacts with myocardin in vivo and in vitro. A, HMG2L1 binds to myocardin family proteins in vivo. Myocardin or HA-tagged MRTF-A/B adenovirus were transduced into A7r5 SMCs. Subsequently, nuclear extract was harvested, and proteins were immunoprecipitated with anti-myocardin, HA, HMG2L1, or control IgG antibodies. The immunoprecipitated proteins were detected by Western blotting using anti-myocardin, anti-HA, and anti-HMG2L1 antibodies, as indicated at the right of the blot. 10% total extract was loaded as input. B, HMG2L1 does not bind to SRF. GST fused to SRF or GST alone was expressed in bacteria, conjugated to glutathione-Sepharose beads, and incubated with bacterially expressed T7 tagged amino-terminal-myocardin (amino acids 1–585) or full-length HMG2L1 as indicated. Bound proteins were identified by Western blotting with an anti-T7 antibody (upper panel). The lower panel showed the expression of the GST-SRF fusion protein or GST alone (marked by an asterisk to the top left of each protein). The interaction between myocardin and SRF served as a positive control. C, schematic representation of myocardin indicating the GST-fusion proteins analyzed and summary of myocardin domains mapped for interacting with HMG2L1 from D and E. NTD, N-terminal domain; ++, basic domain; Q, poly Q domain; LZ, leuzine zipper domain; TAD, transcriptional activation domain; mut: mutant. D, HMG2L1 binds to the full-length myocardin. Bacterially expressed full-length myocardin was incubated with the GST-HMG2L1 fusion protein. Western blotting was performed to detect the myocardin fusion protein that bound to HMG2L1 (upper panel). The lower panel in D indicates the expression of the GST fusion protein of full-length HMG2L1 as detected by a Ponceau S staining. E, HMG2L1 binds to the myocardin N-terminal domain, basic, and poly Q domain. Bacterially expressed HMG2L1 was incubated with the series of GST-myocardin fusion proteins indicated in C. Western blotting was performed to detect the GST-myocardin fusion proteins that bound HMG2L1 (upper panel). The lower panel in E indicates the expression of the myocardin GST fusion proteins as detected by a Ponceau S staining. *, GST fusion protein.