Mouse monoclonal anti myc antibody

The human HATL5 serine protease (SP) domain was cloned into the pSecTag2a plasmid (Life Technologies, Grand Island, NY) for expression and analysis in mammalian cell culture. HATL5 serine protease domain sequence from the human full-length HATL5-V5-His plasmid was PCR amplified using the following primers: 5′-CCAAGGATCCATGTTGTGGGAGACAAGTAGCCAAC-3′ and 5′-CCAAGCGGCCGCGAGTCCAGTCTTGGATGTAATCC-3′. The resulting PCR fragment was cloned into the pSecTag2a vector between the BamHI and NotI sites using standard techniques. Transfection of CHO cells (ATCC, Manassas, VA), protein extraction, and western blot analysis was performed as described above. A mouse monoclonal anti-Myc antibody (Invitrogen, Life Technologies, Grand Island, NY) was used for detection by western blotting.

Total protein was extracted from cells using extraction solution (50 mM Tris pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP40 and 1 mM DTT) supplemented with protease inhibitors (Thermo Fisher Scientific, Inc.). Samples were incubated on ice for five minutes followed by centrifugation (1700 rpm, 4 °C, 5 min). Protein concentrations were determined using the Bradford method (Pierce Coomassie Protein Assay Kit, Life Technologies). Samples (20 μg protein/lane) were separated on 10–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Amersham). After blocking in PBS-Tween 0.1% buffer containing 5% non-fat milk, membranes were first incubated with mouse monoclonal anti-MYC antibody (diluted 1:5000, Invitrogen), Cyclin B1 (diluted 1:10000, Abcam) or mouse monoclonal anti-VCP antibody (diluted 1:7000, BD Biosciences) for 2 h following incubation with goat polyclonal anti-mouse Immunoglobulins/HRP secondary antibodies (diluted 1:7000, Dako) for one hour. Subsequently, blots were imaged using an enhanced chemiluminescence kit (Amersham).

CoIPs were performed as previously described (Nelson et al., 2016 (link)). Briefly, HEK293 cells were co-transfected with expression plasmids encoding Myc-SERCA2a and HA-PLN in the presence of increasing concentrations of HA-DWORF or control plasmid. Whole cell lysates were prepared in CoIP buffer (20 mM NaPO₄, 150 mM NaCl, 2 mM MgCl₂, 0.1% NP-40, 10% Glycerol, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM DTT and Complete protease inhibitor [Roche, Basel, Switzerland]). Immunoprecipitations were carried out using 1 mg of mouse monoclonal anti-Myc antibody (Invitrogen, Carlsbad, CA) and collected with Dynabeads (Invitrogen, Carlsbad, CA). Tris/Tricine gel electrophoresis was performed using pre-cast 16.5% Mini-PROTEAN Tris-Tricine gels (Bio-Rad, Hercules, CA). Standard western blot procedures were performed on input and IP fractions using the following antibodies: HA (Invitrogen, Carlsbad, CA), Myc (Invitrogen, Carlsbad, CA) or GAPDH (Invitrogen, Carlsbad, CA). Specific catalogue numbers for the reagents used for these CoIP studies can be found in the Key Resource Table.