rhPRG4 was incubated with β1-3 galactosidase and α2-3, 6, 8 neuraminidase (New England BioLabs, Ipswich, MA, US) separately or in combination (simultaneously) according to the manufacturer’s instructions. FLSs were prepared (Ren et al., 2021 (link)) from normal individuals (n = 3) and OA patients (n = 3) and were incubated with 15 mg of rhPRG4 (intact, deglycosylated with α2-3, 6, 8 neuraminidase, β1-3 galactosidase, or both) for 48 h at 37°C at 5% CO2. The effect of the deglycosylation was monitored as a shift in the migration of rhPRG4 using SDS-PAGE (Supplementary Figure S3).
Huang S., Thomsson K.A., Jin C., Ryberg H., Das N., Struglics A., Rolfson O., Björkman L.I., Eisler T., Schmidt T.A., Jay G.D., Krawetz R, & Karlsson N.G. (2022). Truncated lubricin glycans in osteoarthritis stimulate the synoviocyte secretion of VEGFA, IL-8, and MIP-1α: Interplay between O-linked glycosylation and inflammatory cytokines. Frontiers in Molecular Biosciences, 9, 942406.
Corresponding Organization : OsloMet – Oslo Metropolitan University
Other organizations :
Sahlgrenska University Hospital, University of Calgary, Alberta Bone and Joint Health Institute, Lund University, Karolinska Institutet, UConn Health, Providence College, Brown University
Incubation of rhPRG4 with α2-3, 6, 8 neuraminidase
Incubation of rhPRG4 with both β1-3 galactosidase and α2-3, 6, 8 neuraminidase (simultaneously)
dependent variables
Effect of deglycosylation on the migration of rhPRG4 using SDS-PAGE
control variables
Incubation time (48 h)
Incubation temperature (37°C)
Incubation atmosphere (5% CO2)
FLSs from normal individuals (n = 3) and OA patients (n = 3)
controls
Intact rhPRG4 (no deglycosylation)
Annotations
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