Fig 2: MARCKSL1 contributes to cellular signaling that regulates NCAM1 polysialylation.(a) PrP-deficient NMuMG cells undergoing EMT exhibit reduced levels of membrane-associated MARCKSL1. Western blot analysis of cellular fractions from wild-type (wt) or PrP knockout (ko) NMuMG cells before or after exposure to TGFB1 for 11 h. (b) TGFB1-treated NMuMG cells deficient for MARCKSL1 exhibit a significant upregulation of steady-state protein levels of PrP and NCAM1 but fail to promote NCAM1 polysialylation. (c) Graphs depicting intensity quantitations of protein bands shown in subpanel ‘b’. Asterisks indicate significant protein level (MARCKSL1 and NCAM1) or protein ratio differences (PrP / actin ratios and total NCAM1 / non-polysialylated NCAM1) in direct comparisons (p-value < 0.05). (d) MARCKSL1 deficiency impairs ST8SIA2 transcription following 48 h TGFB1 treatment of NMuMG cells. RT-PCR-based relative quantitation of ST8SIA2 transcript levels (based on oligo dT or random primers) in TGFB1-treated NMuMG cells deficient for MARCKS or MACKSL1. Data were normalized relative to vehicle-treated control cells.

Fig 3: Cartoon depicting MARCKSL1 in a PrP-dependent signaling loop that controls NCAM1 polysialylation in NMuMG cells undergoing EMT.Note that evidence for PrP controlling St8sia2 gene expression had been previously established [8].

Fig 4: PrP-deficiency causes pronounced and consistent changes to steady-state levels of members of MARCKS protein family.(a) Inverse effect of PrP-deficiency on the relative abundance ratios of MARCKS and MARCKSL1. (b) Sequence alignment of MARCKS and MARCKSL1. (c) Charts depicting the inverse distributions of TMT ratios for peptides assigned to MARCKS and MARCKSL1. Although relative enrichment levels (depicted as log2 ratios) of MARCKS (or MARCKSL1) varied from cell model to cell model, in all cell models tested peptide mixtures obtained from wt cells (labeled with even-numbered TMT reagents) comprised higher (or lower) relative levels of MARCKS (MARCKSL1)-derived peptides than the corresponding peptide mixtures from PrP-deficient cells (labeled with odd-numbered TMT reagents). Note the different ordinate scales on individual plots. The blue dots represent signature ion mass spectra selected for quantitation while the red dots represent those excluded. The largely overlapping distribution of blue and red dots is the consequence of a data processing configuration that relied on two complementary algorithms, Mascot and Sequest, for the assignment of spectra to peptides. To avoid artificial inflation of the number of peptides identified and quantified, the program identifies these occurrences as duplicate hits and rejects one of the replicates when tallying quantified peptides for a given protein. (d) Validation of steady-state expression level changes of MARCKS and MARCKSL1 by Western blot analyses. The even distribution of actin bands across all samples verified successful adjustment of equal protein content by BCA assay. Asterisks designate the presence of a cross-reactive band labeled with the MARCKSL1 antibody that served as an additional internal loading control.