Journal: The Journal of Biological Chemistry
Article Title: Sal-like 4 protein levels in breast cancer cells are post-translationally down-regulated by tripartite motif–containing 21
Figure Lengend Snippet: TRIM21 targets Lys-190 in SALL4 amino acid sequence. A , structures of fragmented-SALL4B-EGFP probes are shown. NLS , nuclear localization signal. B , EGFP signals of MCF-7 cells expressing the probes are shown ( n = 3). TRIM21 knockdown was performed with shTRIM21-5. The identified TRIM21 target region (aa 172–255) is depicted. C , the immunoblot image shows GFP immunoreactions of MCF-7 cells expressing SALL4B(1–255)-EGFP probes with Lys to Arg mutations ( mut. ) at residues 173, 175, and 190 ( n = 3). D , TRIM21 knockdown was performed in MCF-7 cells expressing the SALL4B(1–255) K190R-EGFP probe ( n = 3). E , the immunoblot image shows the signal of the polyubiquitinated SALL4B(1–255)-EGFP probe ( n = 3). MCF-7 cells were treated with MG-132. Input samples were used for the total amount of each probe (α-GFP) and internal control (α-β-actin). F , the immunoblot image shows overexpression of the SALL4B-EGFP probe with or without K190R mutation ( n = 3). Short- and long-exposure images of GFP immunoreaction are shown. The results of migration assays are shown. Arrowheads indicate migrated cells. Scale bar , 100 μm. The graph shows the migrated cell numbers. n.s. , not significant; *, p
Article Snippet: The primary antibodies were anti-SALL4 antibody (ab29112, lot number GR146101-1, Abcam; 1:1000 dilution), anti-GFP antibody (ab290, lot number GR19413-1, Abcam; 1:1000 dilution; validated by immunoblotting in EGFP-overexpressing cells), anti-SALL1 antibody (AP17204a, lot number SA111125AA, Abgent, San Diego, CA; 1:1000 dilution; validated by immunoblotting in TRIM21 knockdown cells), and anti-β-actin antibody (ab6276, lot number 66278-2, Abcam; 1:5000 dilution).
Techniques: Sequencing, Expressing, Over Expression, Mutagenesis, Migration