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. 2021 Mar 4;41(3):BSR20202828. doi: 10.1042/BSR20202828

Figure 2. Confirming MAK3 deletion and HA-NAA30 expression.

Figure 2

(A) Deletion of MAK3 was achieved by homologous recombination by replacing the MAK3 ORF with a KanMX cassette conferring geneticine resistance in yeast. Gene disruption of MAK3 in the mak3∆ deletion strains was confirmed by colony PCR using a primer pair that was positioned 300–450 bp from the start and stop codon of MAK3. MAK3 PCR product 1303 bp (MAK3 531 bp + 772 bp flanking region) and MAK3::kanMX6 PCR product 2128 bp (KanMX6 1356 bp + 772 bp flanking region). (B) Expression of HA-NAA30 was confirmed by immunoblotting using anti-HA. Actin and stain-free imaging served as loading controls. Asterisk (*) indicates unspecific band in the W303-1A strain background. Full immunoblot images shown in Supplementary Figure S1.