Figure 4 | Oncogene

Figure 4

From: The FHA domain protein SNIP1 is a regulator of the cell cycle and cyclin D1 expression

Figure 4

SNIP1 associates with BRG1 and not p300 in U-2 OS and HeLa cells. (a) Association of SNIP1 with p300 is cell type specific. Endogenous SNIP1 was immunoprecipitated from 200 μg of NMuMg cells, U-2 OS cell whole-cell extracts or 1 mg of HeLa cell nuclear protein extract. The immunoprecipitated complex was resolved by SDS–PAGE and immunoblotted for SNIP1 and p300. Input lanes represent 10, 5 and 1% of total protein from NMuMg, U-2 OS and HeLa extract used in each immunoprecipitation, respectively. Preimmune serum from the rabbit used to raise the SNIP1 antibody was used as a negative control. (b) SNIP1 is found in a high molecular weight complex. HeLa nuclear protein extract was resolved by Superose 6 gel filtration. Fractions (50 μl) were collected and resolved by SDS–PAGE followed by immunoblotting for SNIP1, p300 and BRG1. Input lanes represent 5% of the HeLa nuclear extract used in the gel filtration analysis. SNIP1 was not detected in any fraction containing lower molecular weight complexes. The positions of molecular weight markers used during column calibration are shown. (c) SNIP1 coimmunoprecipitates with BRG1. Endogenous SNIP1 was immunoprecipitated from 200 μg of HeLa cell nuclear protein extract. The immunoprecipitated complex was resolved by SDS–PAGE and immunoblotted for SNIP1 and BRG1. Input lanes represent 10% of total protein from HeLa extract used. Preimmune serum from the rabbit used to raise the SNIP1 antibody was used as a negative control. (d) Colocalization of transfected SNIP1 and BRG1. U-2 OS cells were transfected with YFP-SNIP1 and GFP-BRG1 expression plasmids. At 48 h post transfection, cells were analysed and images were acquired using a DeltaVision microscope. Images were deconvoluted using SoftWorx (Applied Precision). DAPI stain was used to stain the cellular DNA. The yellow colour in the merged image indicates colocalization of YFP-SNIP1 with GFP-BRG1. (e) Characterization of the 2B12 SNIP1 monoclonal antibody. SNIP1 monoclonal antibody (clone 2B12) was tested for specificity by immunoblotting 100 μg of Hela nuclear extract. (f) Endogenous SNIP1 partially colocalizes with BRG1 and BRM. U-2 OS cells were permeabilized and stained with SNIP1 (2B12) (red), BRG1 (green) and BRM (green) antibodies. DAPI stain was used to stain cellular DNA. Image analysis was performed as in (d). Since these are digitalized images, both BRG1 and BRM signals are set to green even though they are from the same cell, using separate secondary antibodies. The yellow colour in the merged image indicates colocalization

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