V5 tag mouse monoclonal antibody

Proteins were separated by SDS-PAGE, transferred to a PVDF membrane (BioRad) and immunoblotted with primary monoclonal anti-FLAG M2 antibody (Sigma-Aldrich, F3165, diluted 1:5000); V5 tag mouse monoclonal antibody (Thermo Fisher Scientific, R960-25, diluted 1:5000), Pgk1 monoclonal mouse antibody (Invitrogen, 22C5D8, diluted 1:10,000) and HRP-conjugated secondary antibodies, detected with Femto maximum sensitivity substrate (Thermo Fisher Scientific). ImageLab software (Bio-Rad) was used for image acquisition and densitometric analysis. The density of a given band was measured as the total volume under the peak and corrected with background subtraction. The intensity was normalised with respect to corresponding Pgk1 signals of each sample.

Shortly before the DL2-B2 cells were used in experiments, immunohistochemistry was performed to test the expression of the vector (S1 Fig). pMT contains the V5 epitope tag allowing its rapid detection with Anti-V5 Antibody. DL2-B2 induced and uninduced cells (CuSO4-free) were cultured under the conditions mentioned before. Briefly, 1 ml of medium was centrifuged at 2000g for 3 min, the supernatant medium was discarded and washed twice with 1% v/v phosphate-buffered saline (PBS). Then, 20 μl of the cells were placed on a slide coated with Poly-L-Lysine for 30 min. Every well was washed 3x with 1% v/v PBS, and 20 μl of 4% w/v paraformaldehyde was added for 30 min, followed by blocking and permeabilization with 1× PBS, 0.01% v/v Triton-X (Sigma), 1% v/v Normal Goat Serum (NGS) for 30 minutes. The cells were labelled with V5 tag mouse monoclonal antibody (Thermofisher) at a dilution of 1:1000 in 1% v/v PBS and incubated overnight at 4°C, then labelled with Alexa Fluor 488 rabbit anti-mouse IgG secondary antibody (Thermofisher) at a dilution of 1:1000 and incubated overnight at 4°C. Finally, cells were washed 3x with 1% PBS and stained with Hoechst for 5 min and washed 3x with PBS. The slide was mounted with 80% glycerol and edges were sealed with nail polisher. The images were taken on a Nikon Eclipse 90i microscope at 20× magnification and processed using the Fiji software.

Plasmids were constructed to encode the four different NAA10 (NM_003491.4) variants; NAA10 c.16G>C p.(A6P), NAA10 c.235C>T p.(R79C), NAA10 c.386A>C p.(Q129P), and NAA10 c.469G>A p.(E157K). The mutations were incorporated into a pcDNA3.1/NAA10-WT-V5 vector [previously called phARD1-V5 (Arnesen et al. 2005 (link))] using Q5 Site Directed Mutagenesis Kit (New England Biolabs). The primers used for mutagenesis are available in Supplementary Table S3. The resulting plasmids were verified by Sanger sequencing. Primary antibodies used in this study were V5-tag mouse monoclonal antibody (1:5000 dilution, Invitrogen R960-25), β-tubulin mouse monoclonal antibody (1:3000 dilution, Sigma-Aldrich T5293) and NAA15 rabbit polyclonal antibody (1:2000, Biogenes, previously called anti-NATH) (Arnesen et al. 2005 (link)). Secondary antibodies used were Mouse IgG HRP-Linked Whole Ab (1:5000 dilution, Cytiva NA931) and Rabbit IgG HRP-Linked Whole Ab (1:5000 dilution, Cytiva NA934).