Fig. 3.

N-terminome analysis of CM of SPPL3-deficient isogenic HeLa cells. a PCR genotyping of the exon 8 region of the SPPL3 locus in HeLa cells following Cas9 RNP electroporation. The parental 545 bp band was only detected in cells treated with sgRNAs directed against a control target locus. It was diminished in pooled cells following SPPL3 targeting and completely absent from SPPL3 KO clones E5, F6, F7. Truncated PCR products demonstrate deletions within exon 8. b SPPL3 immunoblotting of carbonate-washed membranes obtained from SPPL3 KO clones and parental HeLa cells (ctrl). c Detection of MGAT5 and B4GALT1 in CM and cellular membranes from SPPL3 KO clones and parental HeLa cells. TCA-precipitated conditioned supernatants (left) and lysates of carbonate-washed cellular membranes (right) were subjected to immunoblotting with the indicated antibodies. Calnexin was used as a loading control for the membrane fraction. d Volcano plots showing peptides detected following HYTANE enrichment of CM from SPPL3-deficient clones and parental HeLa cells. Samples were analysed in parallel on the indicated LC–MS systems. 7864 and 5372 unique peptides are plotted, respectively and labelling/filtering is performed as outlined in Fig. 2e with x = 50 aa. Dashed lines represent threshold values (± 2 SD for log2 difference and p = 0.05). e Schematic overview and mapping of neo-N peptides found to be changed in abundance in CM (see Fig. 2h)