MYC Epitope Tag Antibody

Anti-Myc epitope tag polyclonal antibody detects ~ 100 kDa CARBOXY terminal linked Myc-tagged recombinant protein present in ~35 µg of lysate by western blot.

Anti-Myc epitope tag polyclonal antibody detects both AMINO and CARBOXY terminal linked Myc-tagged recombinant proteins by western blot. Polyclonal rabbit host anti-Myc epitope tag antibody was diluted to 1.0 µg/ml to detect either recombinant protein. 4-20% gradient gels were used to resolve the proteins by SDS-PAGE. The proteins were transferred to nitrocellulose using standard methods. After blocking, the membranes were probed with the primary antibody overnight at 4°C followed by washes and reaction with a 1:10000 dilution of IRDye® 800 conjugated Gt-a-Rabbit IgG (H&L) MX10 for 45 min at room temperature (Green, 800 nm channel). Pre-stained molecular weight markers are also shown (lane M, Red, 700 nm channel).

Git1 phosphorylation at Tyr-554 was enhanced by co-expression of paxillin. A, Western blotting of protein expression levels in HEK293T cells exogenously expressing the FLAG-tagged Git1-Y9F-Y554 mutant together with Myc-tagged paxillin, Myc-tagged Hic-5, or a control mock. B, Tyrosine phosphorylation of Y9F-Git1 proteins in anti-FLAG immunoprecipitates. The lower graph shows the densitometric analysis of the Western blotting data. Data are the mean ± S.E. (error bars; n = 3). *, P < 0.05 significantly different from Hic-5-transfected cells by ANOVA with Fisher's PLSD post hoc tests.

Git1 phosphorylation at Tyr-554 weakened its association with Hic-5. A, Western blotting of protein expression levels, and tyrosine phosphorylation of all proteins in HEK293T cells expressing FLAG-tagged Git1 proteins (Fig. 1A) together with Myc-tagged Hic-5. Cells were treated with 100 µm pervanadate or vehicle for 15 min, and then analyzed by Western blotting using anti-FLAG M2, anti-Myc 9E10, or anti-phosphotyrosine PY20. B, Co-immunoprecipitaion of Git1 mutants with Hic-5. The immunoprecipitates from cell extracts with anti-FLAG beads were analyzed by Western blotting with an anti-FLAG or anti-Myc antibody. To verify the tyrosine phosphorylation of FLAG-tagged Git1 proteins, the same membrane was reacted with anti-phosphotyrosine PY20. Ig, immunoglobulin. The lower part shows the densitometric analysis of the relative amount of Myc-Hic-5 to FLAG-Git1 in the immunoprecipitates. Data are the mean ± S.E. (error bars; n = 3). **, P < 0.01 significantly different from the wild-type with the same treatment; #, P < 0.05 or ##, P < 0.01 significant difference between vehicle- and pervanadate-treated groups by ANOVA with Fisher's PLSD post hoc tests.

SARS 3a induces NLRP3 inflammasome activation by multiple mechanisms. A) Immunoblot analysis of the pro- and cleaved forms of caspase-1 and IL-1β after reconstitution of inflammasome in HEK 293T cells transfected with SARS 3a with or without NEK7 shRNA. B) Immunoblot analysis of the pro- and cleaved forms of caspase-1 and IL-1β after reconstitution of inflammasome and transfection with SARS 3a or SARS 3a C133A. C) Immunoblot analysis of the pro- and cleaved forms of caspase-1 and IL-1β after co-transfection with caspase-1, IL-1β, and SARS 3a or SARS 3a C133A. D) Immunoprecipitation analysis of interaction between SARS 3a or SARS 3a C133A and caspase-1. All western blot data are representative of two or three independent experiments.