Fig. 3

TRIM22 does not interact with Sp1 and does not alter Sp1 expression and phosphorylation. a 293T cells were transfected with 1.5, 3, 4.5, or 6 µg of a TRIM22-expressing plasmid, equalizing the DNA doses with empty pcDNA3.1(+) vector. 48 h post-transfection, whole cell extract (WCE) was analyzed by western blotting with anti-Sp1, anti-TRIM22 and anti-Actin antibodies (Abs). A similar level of Sp1 was observed in the mock-treated cells, control cells (empty pcDNA3.1 only) and cells expressing increasing concentration of TRIM22 expressing plasmid. One representative of three independent experiments is shown. b 293T cells were transfected with 6 µg of either a TRIM22-expressing plasmid (T22) or pcDNA3.1(+) plasmid (ctrl). 48 h post-transfection, WCE was prepared in the presence of PhosStop phosphatase inhibitor cocktail (Roche). An aliquot of 50 µg was treated with 5 I.U. of SAP (Roche) for 45 min at 37 °C and subjected to western blot analysis. The level of phosphorylated Sp1 was not altered by TRIM22 expression (lanes 2 and 3). SAP treatment caused the disappearance of the phosphorylated forms of Sp1 (upper band), without affecting overall Sp1 levels detected between TRIM22-expressing and control conditions (lanes 5 and 6). One representative of two independent experiments is shown. c 293T cells were transfected with either TRIM22-expressing or Luc-expressing plasmid as unrelated control protein. 24 h post-transfection, 90 % of WCE was immunoprecipitated 2 h at 4 °C with anti-TRIM22 (Abnova) Ab, whereas a 10 % volume was saved as input fraction. Immunocomplexes were captured using magnetic Dynabeads protein G (Life Technologies), according to the manufacturer’s protocol. Western blotting was performed with anti-Sp1 (Millipore), anti-Firefly Luciferase (Millipore) and anti-Actin Abs. Endogenous Sp1 did not immunoprecipitate with TRIM22. One representative of two independent experiments is shown