|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
Fig. 9. Barx2 and Sox9 bind to endogenous Col2a1 enhancer during
chondrogenic differentiation. (A) Crosslinked chromatin from limb mesenchymal
cells and C3H10T1/2 was precipitated with Barx2 or nonspecific antibody and
analyzed by PCR using primers spanning the Col2a1 enhancer. Lanes
1-6, amplification of Col2a1 enhancer: 1, no Barx2 antibody added; 2,
amplification of Col2a1 enhancer from input DNA; 3,
immunoprecipitation with non-specific antibody; 4, immunoprecipitation with
Barx2 antibody; 5, immunoprecipitation with normal rabbit IgG; 6,
immunoprecipitation with Barx2 antibody. (B) Barx2 has stronger association
with Col2a1 enhancer after the BMP treatment. Equivalent amount of
crosslinked chromatin from treated with BMP and untreated limb mesenchymal
cells were immunoprecipitated with Barx2 antibody and analyzed by PCR using
primers spanning Col2a1 regulatory region. Gapdh control
confirming that equivalent amounts of chromatin were used in each ChIP assay.
(C,D) During chondrogenic differentiation the Col2a1 enhancer is
occupied by both Barx2 (C) and Sox9 (D). D1 cells were simultaneously
transfected with Myc-tagged Barx2 and Flag-tagged Sox9 expression vectors and
cultured under differentiation conditions. Approximately equivalent amounts of
crosslinked chromatin were immunoprecipitated in parallel with antibodies
specific for Myc, flag, Phospho-Sox9 or normal rabbit IgG, and Col2a1
enhancer region was amplified by PCR. (C) Lane 1, no antibody; 2, input DNA;
3, rabbit IgG; 4, Myc antibody. (D) Lane 1, negative control; 2,
immunoprecipitation with Flag antibody; 3, with Phospho-Sox9 antibody; 4,
positive control, input DNA. Arrows indicate Col2a1 product.
|
