Anti myc antibody 9e10

HEK-293 cells were lysed on ice for 45 min in ice cold 150 mM NaCl, 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100 (v/v), 50 mM Tris pH 7.5 (IP buffer) supplemented with 5 mM PMSF, 1 mM vanadate and 1:100 of proteases inhibitor cocktail (P8340, Sigma). Cells were removed with a scraper, homogenized and centrifuged at 16000 g for 20 min at 4°C. For anti-myc IP, the supernatant was incubated at 4°C for 1–2 h with anti-myc 9E10 antibody (Santa-Cruz) followed by a 1.5 h incubation with protein G coupled to sepharose-beads. For anti-HA IP, the supernatant was incubated for 2 h with the anti-HA antibody coupled to agarose beads (Sigma) at 4 C. Immunoprecipitates were then washed at 4°C for a minimum of 5 times with 1 mL of the IP buffer with gentle centrifugations of maximum 400 g. Beads were then resuspended in a Laemmli buffer supplemented with 1% ß-mercapto-ethanol (v/v).

Polyclonal antibodies raised against Xenopus Geminin and Xenopus Cdt1 were described previously [5 (link),8 (link)]. Antibodies to mouse MCM4 were kindly supplied by Hisao Masai, and antibodies to Xenopus MCM3 were kindly supplied by Ron Laskey. Anti-Myc 9E10 antibodies (Santa Cruz sc-40), anti-HA F-7 antibodies (Santa Cruz sc-7392), anti-FLAG M2 antibodies (Sigma-Aldrich F1804) and anti-histone H4 K12Ac antibodies (Upstate Biotechnology) were purchased from the manufacturer.

In order to examine whether the proteins exogenously produced in mammalian cells by transfection of each pcDNA3.1-myc-His(A)-derived plasmid induce necrosis, COS-7 cells were seeded in 24-well plate at 7.5 x 10⁴ cells/well and grown overnight. The cells were transfected and the amounts of LDH released into the extracellular medium were measured at 24 hours after transfection. We measured the LDH amount in the medium of uninfected cells as a control and use the value as background. The absorbance value obtained from untransfected cells was subtracted from all of the absorbance values obtained from cells transfected with each plasmid or treated by Triton X-100. The LDH (%) was shown as a ratio when the value obtained from the well treated with 0.1% Triton X-100 was set as 100%. In order to examine whether the proteins were exogenously produced in mammalian cells, the transfected cells were also washed with PBS twice and lysed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The samples were incubated at 95°C for 5 minutes and then subjected to SDS-PAGE with 12% gel. The proteins in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane and the Myc-tagged proteins were detected by Western blot with anti-Myc 9E10 antibodies (Santa Cruz).

HEK293 cells were transiently transfected with NH₂-terminal Myc-epitope tagged DUOXA2 expression vectors. Cell lysate was then run on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (Thermo Fisher Scientific), and expression of DUOXA2 was analyzed by Western blotting using anti-Myc antibody (9E10; Santa Cruz Biotechnology, Dallas, TX; see Supplementary Data for further details).

Myc-nedd8 and GFP were subcloned into Psp64 poly (A) vector (Promega). AmpliCap SP6 High Yield message maker kit (Epicenter) was used for capped mRNA synthesis. Myc-nedd8 and GFP mRNA were synthesized and injected into zebrafish embryos at one-cell stage (400 pg/per embryo). To confirm expression of injected mRNAs, the embryos injected with Myc-nedd8 mRNA for 3 days were harvested and the expression of Myc-nedd8 was confirmed by Western blot using anti-Myc antibody (9E10, Santa Cruz).

At 40 to 44 h after transfection, HEK293T cells expressing myc-tagged BmGr9 were subjected to immunostaining for BmGr9. For permeabilization, cells were fixed with 4% paraformaldehyde in PBS for 10 min and then permeabilized with 0.1% Triton X100 in PBS for 5 min. After washing with PBS, the cells were incubated with anti-myc antibody (9E10; Santa Cruz Biotechnology), washed, incubated with anti-mouse IgG Alexa Fluor 488 conjugated secondary antibody (Thermo Fisher Scientific). For nonpermeabilization, cells were incubated with anti-myc antibody in staining solution (DMEM supplemented with 5% fetal bovine serum and 1 M Hepes (pH 7.4) to produce a final concentration of 10 mM) on ice for 1 h. After washing the cells with Ringer’s solution (140 mM NaCl, 5.6 mM KCl, 2.0 mM MgCl₂, 2.0 mM CaCl₂, 1.25 mM KH₂PO₄, 9.4 mM glucose, 2.0 mM sodium pyruvate, and 5 mM Hepes; pH 7.4), cells were further incubated with the secondary antibody for 1 h, followed by washing with PBS and fixation with 1% paraformaldehyde in PBS. Samples were observed by a fluorescence microscopy using a model IX73 microscope (Olympus).