Purification of DEF-1 protein. (A) Calf brain lysates were passed over the GST, Src SH3, or Src SH3SH2 affinity column, and associated proteins were electrophoresed on a 10% polyacrylamide gel and visualized by silver stain. Molecular size markers in kilodaltons are indicated on the left. (B) Bound proteins from panel A were eluted with free glutathione and passed over an ATP-agarose column. The associated proteins were electrophoresed on a 5% polyacrylamide gel and visualized by silver staining.

Sequence of DEF-1. (A) Eight unique, overlapping clones were used to obtain the DEF-1 composite sequence. One clone (R19) lacked the sequence SRR at amino acids 304 to 306. The sequence from other clones and an apparent human DEF-1 partial sequence in the EST database suggested that the SRR sequence is an alternative exon (data not shown). The number of the last amino acid in a line is noted on the right. Key: overline, peptide sequenced; underline, putative alternative exon; aqua, pleckstrin homology domain (amino acids 326 to 419); purple, zinc finger (amino acids 457 to 480); red, C2 domain (amino acids 498 to 557); yellow, ankyrin-related motifs (amino acids 604 to 623, 640 to 659, 673 to 692); green, SH3 consensus binding sequences (amino acids 791 to 800, 803 to 809, 828 to 835, 895 to 901, 993 to 999); brown, proline-rich repeat (amino acids 934 to 1001); blue, SH3 domain (amino acids 1074 to 1123). (B) Putative structure of proline-rich motif in DEF-1. The sequence of amino acids 976 to 1001 is drawn in a left-handed poly-proline type II helix. Standard amino acid abbreviations are used.

DEF-1 is an SH3 binding protein. (A) Lysates made with NP-40 lysis buffer from NIH 3T3 cells expressing pLNSL7 alone (pLN) (38) or pLNSL7/HA-tagged DEF-1 (pLN/DEF-1 HA) were passed over the noted columns. Bound proteins were immunoblotted with an anti-HA antibody. (B) Bovine brain (brain extract) or Sf9 cells infected with a pp60^c-src baculovirus (Bv Src) was lysed with NP-40 lysis buffer and passed over a column consisting of GST alone, GST-DEF-1-C₁ (amino acids 777 to 926; SH3 binding sites), or GST-DEF-1-C₂₊₃ (amino acids 928 to 1129; proline-rich repeat plus SH3 domain). Bound proteins were immunoblotted with an anti-pp60^c-src antibody. (C) Bovine brain extracts were passed over a GST-DEF-1-C₁, C₂ (amino acids 934 to 1036; proline-rich repeat) or C₃ column (amino acids 1074 to 1129; SH3 domain), and bound proteins were blotted for DEF-1 by using an anti-DEF-1 rabbit polyclonal serum. The brain lysate was immunoprecipitated with preimmune (pre-imm.) or anti-DEF-1 serum (αDEF-1) or passed over a column of GST alone (GST) as a control.

DEF-1 induces fibroblastic differentiation. (A) Stably infected DEF-1 BALB/c 3T3 cells were grown to confluence and photographed with a 20× objective after noticeable lipid accumulation. (B) NIH 3T3 cells stably infected with DEF-1 or the expression vector alone were grown to confluence with 10% FCS-DMEM and supplemented with combinations of dexamethasone (Dex), insulin, and pioglitazone (Pio) as indicated. After notable lipid accumulation (9 days), the cells were fixed and lipid droplets were identified by staining with Oil-Red-O. (C) Twenty micrograms of total RNA isolated from the cells treated as shown in panel B was probed with an aP2 cDNA (D, dexamethasone; P, pioglitazone; I, insulin; 0, no treatment) (61). (D) 3T3 F442A cells ectopically expressing DEF-1 or the expression vector alone were grown to confluence and supplemented with 10 nM pioglitazone. After 9 days, the cells were stained with Oil-Red-O and photographed.

Expression of PPARγ in DEF-1 cells. RNA isolated from NIH 3T3 cells expressing the vector alone (vector) or DEF-1 (DEF-1) treated as in Fig. 4B was probed with a PPARγ cDNA (60). Quantities of RNA were standardized with a 36B4 probe (30).

DEF-1 expression in various tissues and adipose tissue from mouse models of obesity. (A) The full-length DEF-1 cDNA was used to probe a mouse multiple-tissue Northern blot (Clontech), and 5 μg of poly(A)⁺ mRNA was isolated from mouse adipose tissue. The lower band appears to be smaller than the DEF-1 composite cDNA isolated and, therefore, is believed to be a DEF-1-related mRNA (data not shown). A β-actin probe was subsequently used to normalize mRNA loading. (B) Lysates from 3T3 F442A cells before (Undiff.) and after (Diff.) differentiation with 1 μM pioglitazone were immunoprecipitated with preimmune (PI) or anti-DEF-1 antiserum (αDEF-1). The immunoprecipitates were immunoblotted with the anti-DEF-1 antibody. (C) An example of the data obtained from a competitive PCR analysis used to evaluate the levels of DEF-1 mRNA in adipose and brain tissues isolated from wild-type, ob/ob, and db/db mice. The amount of competitor added that resulted in equivalent DEF-1 and competitor signal intensities was determined. (D) The average fold increase of DEF-1 mRNA levels in adipose tissue isolated from two ob/ob mice and one db/db mouse relative to that of a wild-type standard was determined as described in panel C. A similar analysis was performed with RNA from brain tissue. All values represent the average from three separate trials, and the error bars refer to average deviations.

The C terminus of DEF-1 promotes adipogenesis. NIH 3T3 cells stably expressing DEF-1–Bgl were assayed as described in the legend for Fig. 4B in the presence of dexamethasone, insulin, and pioglitazone.