TRIM68 interaction with TRK-fused gene protein (TFG) increases post TLR3 stimulation.

<p>(A) TRIM68 bound proteins were immunoprecipitated from lysates with anti-FLAG coated agarose beads (Sigma). Immunoprecipitation using normal mouse IgG functioned as a negative control. Selected bands were excised, trypsin digested and analysed by mass spectrometry. A western blot showing corresponding lysates from the silver stained gel was immunoblotted for FLAG to verify successful transfection and immunoprecipitation of FLAG-TRIM68. Immunoblots shown are from a single experiment and are representative of two independent experiments. (B) HEK293T cells were transfected with 2 µg of EV and FLAG-TRIM68. TRIM68 containing complexes were immunoprecipitated using anti-FLAG coated beads and blotted for TFG and FLAG. Lysates were immunoblotted for TFG and FLAG. Immunoblots shown are from a single experiment and are representative of three independent experiments. (C) HEK293T cells were transfected with 2 µg of EV and FLAG-TRIM68. TFG containing complexes were immunoprecipitated using anti-TFG coated beads and immunoblotted for FLAG and TFG. Lysates were immunoblotted for TFG and FLAG. Immunoblots shown are from a single experiment and are representative of three independent experiments. (D) HeLa cells were transfected with 2 µg of GFP-TRIM68 (green) expression plasmid and stimulated with 20 µg/ml of poly(I:C) for 6 and 24 hr. Cells were fixed and then stained with anti-TFG before mounting in DAPI in order to visualise nuclei (blue). Images were taken under oil immersion at 63X magnification. Panels on the right contain zoomed images of white box in previous panel. Images shown are from a single experiment and are representative of three independent experiments. The raw integrated density value (sum of pixel values) was calculated using ImageJ software (1.48 4-bit Java) for TFG only images for each poly(I:C) time point. Presented data is graphed from the average of sixteen separate images ± S.E.M. *p<0.05 was considered significant.