Figure 4.

Different regulation of STAR proteins in alternative splicing regulation in vitro. (A) Co-expression of SAM68 and SLM1 in vivo. SAM68 and SLM1 proteins are detected coexpressed in a subset of midbrain neurons in the adult mouse brain. Bar, 20 µm. (B) Co-immunoprecipitation of HA-tagged SAM68 and GFP-tagged SAM68, SLM1, or SLM2 from HEK293 cells cotransfected with expression constructs. Immunoprecipitation was performed with the anti-HA antibody and proteins in input or immunoprecipitates were detected with anti-HA or anti-GFP antibodies. (C) Complex formation of SAM68 and SLM1 in brain extracts. Complexes were precipitated with either anti-SAM68 (left) or anti-SLM2 (right) antibodies and precipitates probed with anti-SAM68, anti-SLM1, or anti-SLM2 antibodies. When indicated, lysates were treated with Ribonuclease A before antibody addition to eliminate interactions that depend on integrity of cellular RNAs. (D) Alternative splicing of Nrxn1 and Nrxn2 AS4 was assayed by semi-quantitative RT-PCR. To confirm protein expression levels, the lysates of HEK293 cells expressing GFP-SLM1 or GFP-SLM2 and increasing amounts of GFP-SAM68 were subjected to the immunoblotting. Quantitation of alternative splice isoform choice is displayed on the right.