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. Author manuscript; available in PMC: 2013 May 25.
Published in final edited form as: Cancer Cell. 2012 May 25;21(5):694–708. doi: 10.1016/j.ccr.2012.03.040

Fig 6. CD37 cross-ligation increases FoxO3a-dependent transcription of BIM.

Fig 6

(A) Top, immunoblot of nuclear extracts derived from αFc, trastuzumab+αFc (T) or SMIP-016+αFc (S) treated CLL cells; bottom, quantization of the pAKT/total AKT signals in the immunoblots; (B) Immunoblot analysis of nuclear extracts (NE) and cytoplasmic extracts (CE) from CLL cells stimulated with trastuzumab+αFc (T) or SMIP-016+αFc (S) using FoxO3a antibody. BRG-1 and Tubulin were used to confirm quality of the separations. (C) Schema of the Pgl3-BIM reporter construct that includes the reported FoxO3a binding site (FHRE) in the BIM promoter. (D) Luciferase activity of CLL cells transfected with Pgl3-Basic or Pgl3-BIM reporter constructs along with the Renilla luciferase vector and treated without or with SMIP-016+αFc (S) for 12 hr. All values were corrected for co-transfected renilla activity. Data shown are normalized to trastuzumab+αFc treated cells (n=4). (E) Nuclear extract from trastuzumab+αFc (T) or SMIP-016+αFc (S) treated CLL cells were incubated with 32P-labelled wild-type (WT) BIM probe containing FoxO3a binding site and analyzed by EMSA. Representative of nine patient samples. (F) Nuclear extract from SMIP-016+αFc treated CLL cells were incubated with 32P-labelled wild-type (WT) or mutant (M) BIM probes in the presence or absence of 50x unlabeled wild-type or mutant BIM probes. (G) Biotinylated double-stranded oligonucleotides containing FHRE, coupled to streptavidin agarose beads, were incubated with nuclear extracts from trastuzumab+αFc (T) or SMIP-016+αFc (S) treated CLL cells. Bound proteins were eluted and analyzed by FoxO3a immunoblot. Representative of four patient samples. M=Mutant probe; WT=Wild-type probe. (H) Chromatin from trastuzumab+αFc (T) or SMIP-016+αFc (S) treated CLL cells (12 hr) was analyzed by ChIP assay for binding of FoxO3a to the BIM promoter (n=4). The binding of H3 to the GAPDH promoter (H3 element) was also evaluated as a control as SMIP-016 does not affect GADPH expression. Data are represented as mean ± SD for all the relevant panels. See also Figure S4.