Figure 2

COS-1 cells transfected with plasmids encoding shRNAs specific to BTBD1 and BTBD2 show reductions in the specific mRNAs and cytoplasmic bodies stained with BTBD1-specific antibodies. A. Northern blot shows reduced levels of expression of BTBD1 and BTBD2 mRNA in COS-1 cells transfected with BTBD1 or BTBD2 specific silencer constructs. COS-1 cells were untransfected (Un) or transfected with the negative control vector, pSIREN (S-ve), the pSIREN vector encoding Luciferase silencer (S-luc), pSIREN vector encoding BTBD1 shRNA (D1 #3) or BTBD2 shRNA (D2 #7). 48 hours post-transfection, transiently transfected and untransfected (COS-1) cells were harvested for RNA isolation. The Northern blot was hybridized with BTBD1 and BTBD2 specific probes. Equal amounts of RNA (10 μg) were loaded onto each lane as seen with the β-actin specific probe. B. Representative immunofluorescence images showing cytoplasmic bodies in COS-1 cells transfected with pSIREN negative control vector (S-ve), silencer constructs for BTBD1 or BTBD2, or both BTBD1 and BTBD2. Seventy-two hours post-transfection cells were labeled with rabbit BTBD1-specific peptide antibody (#4358) followed by Alexa Fluor 568 goat anti-rabbit IgG (Molecular Probes). C. Quantification of BTBD1/D2 cytoplasmic bodies. The acquired images were analyzed using TIFFany 3 (Caffeine Software) and the cytoplasmic bodies were quantified using Mathematica software (Wolfram research).