Journal: Nucleic Acids Research

Article Title: Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene

doi: 10.1093/nar/gki897

Figure Lengend Snippet: SRp40 and SRp55 interact with ISE-3 sequence and enhance splicing of exon 3. ( A ) Schematic representation of the constructs carrying wild-type (ISE3wt) and disrupted (ISE3m) ISE of apoA-II intron 3 used for the EMSA. ( B ) EMSA of ISE3wt and ISE3m RNAs incubated with HeLa nuclear extract. Complexes were then fractionated on a 4% non-denaturing polyacrylamide gel. The position of RNA–protein complexes (upper complex—Uc and lower complex—Lc) and free RNA are shown. Addition of nuclear extract is also indicated below the lane numbers ( C ) SDS–PAGE analysis of immunoprecipitation with the specific monoclonal antibody against the phosphorylated RS domain (MAb 1H4) following UV-crosslinking of the labeled RNAs ISE3wt, ISE3m and htot as a positive control (plus) . Specific immunoprecipitation of SRp40 (upper panel) and SRp55 (lower panel) is shown only with ISE3wt and htot RNAs. ( D ) Effects of SRp40 and SRp55 overexpression on apoA-II exon 3 splicing. Relative amounts of exon 3 skipping are indicated below the lane numbers. The variability among three different experiments was always <20%.

Article Snippet: After the 30 min incubation with RNase at 37°C, 150 μl of immunoprecipitation (IP) buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA and 0.25% Nonidet P-40) was added to each sample together with 1 μg of monoclonal antibodies mouse anti-SR 1H4 (Zymed Laboratories Inc., San Francisco, CA) and incubated for 2 h at 4°C on a rotator wheel.

Techniques: Sequencing, Construct, Incubation, SDS Page, Immunoprecipitation, Labeling, Positive Control, Over Expression

Journal: Nucleic Acids Research

Article Title: Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene

doi: 10.1093/nar/gki897

Figure Lengend Snippet: TDP-43 binds to the (GU) repeats in the apoA-II context. ( A ) Diagrammatic representation of minigene constructs used for competition experiments and immunoprecipitation with antibodies against TDP-43. Gray and white boxes represent the apoA-II exon 2 and 3, respectively. Thin lines and black circle correspond to the introns and the (GT) 16 repeats. Complete deletion of the (GT) 16 tract in the construct mgΔ(UG) is indicated by a white cross in the black circle. ( B ) UV-crosslinking with competition analysis following addition of 5- to 10-fold molar excess of cold (GU) 16 -Ex3, mgΔ(GU) and mg(GU) 16 RNAs to labeled mg(GU) 16 RNA in the presence of HeLa nuclear extract. ( C ) UV-crosslinking plus immunoprecipitation with anti-TDP43 serum. [α- 32 P]UTP-labeled mg(GU) 16 and mgΔ(GU) RNAs were incubated with HeLa nuclear extract before UV-crosslinking. Immunoprecipitation was then carried out with equal amounts of each UV-crosslinked sample using a polyclonal antiserum against TDP-43.

Article Snippet: After the 30 min incubation with RNase at 37°C, 150 μl of immunoprecipitation (IP) buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA and 0.25% Nonidet P-40) was added to each sample together with 1 μg of monoclonal antibodies mouse anti-SR 1H4 (Zymed Laboratories Inc., San Francisco, CA) and incubated for 2 h at 4°C on a rotator wheel.

Techniques: Construct, Immunoprecipitation, Labeling, Incubation, Cross-linking Immunoprecipitation