Journal: Scientific Reports
Article Title: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function
Figure Lengend Snippet: Functional validation of the BirA*-Tax fusion protein and identification of p62 as a new candidate partner of Tax. ( a ) Lysates from HEK293T cells transfected with the indicated plasmids for 24 h and then treated overnight with biotin or left untreated were analyzed by western blot. ( b ) U2OS cells transiently expressing Tax-His or Myc-BirA*-Tax-His and treated overnight with biotin were analyzed by epifluorescence microscopy after staining with Streptavidin (Strept., green) and His-specific antibodies (red). Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar = 10 μm. The arrows indicate perinuclear accumulation of Tax reminiscent of the Tax/IKK signalosome. ( c ) HEK293T cells were transfected with Myc-BirA* or Myc-BirA*-Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the “Myc-BirA*” condition. The graph shows the result from a representative experiment. ( d ) Lysates from HEK293T cells transiently expressing Myc-BirA* or Myc-BirA*-Tax-His were submitted to a His-specific Ni-NTA pulldown in denaturing conditions before western blot analyses. WCL, whole cell lysate. ( e ) SQSTM-1/p62 is a BirA*-Tax-specific biotinylated protein identified by mass spectrometry. ( f ) Lysates from HTLV-1 chronically infected cells (C8166, HuT102 or C91PL cells) were immunoprecipitated with a p62-specific or Tax-specific antibody, or with control Ig (IP Ig). Samples were then analyzed by western blot. WCL, whole cell lysates. Full-length blots are presented in Supplementary Fig. S4 .
Article Snippet: Antibodies and reagents The following antibodies were used: anti-His (ab9136 or ab18184, Abcam or sc-804, Santa Cruz), anti-Tax1 (Tab172, NIH), anti-p62 (GP62-C, Progen), anti-OPTN (100000, Cayman), anti-GM130 (610823, BD), anti-IKKγ (611306, BD), anti-phosphorylated IKKα/β (2078, Cell Signaling Technology), anti-IκBα (4814, Cell Signaling Technology), anti-phosphorylated IκBα (9246, Cell Signaling Technology), anti-FLAG (M2, Sigma-Aldrich), anti-Myc (4A6, Millipore), anti-Ub (P4D1, sc-8017, Santa Cruz), anti-actin (AC-74, Sigma-Aldrich), anti-Nup153 (147050002, Covance).
Techniques: Functional Assay, Transfection, Western Blot, Expressing, Epifluorescence Microscopy, Staining, Construct, Luciferase, Activity Assay, Mass Spectrometry, Infection, Immunoprecipitation