Skip to main content
. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Cell Mol Life Sci. 2019 Mar 27;76(17):3449–3464. doi: 10.1007/s00018-019-03081-w

Figure 3.

Figure 3.

VC induced N259-linked glycosylation on P4HA1. (A) Treatment of MEF cells for 6 hrs with VC concentration of ≥40 μM induced expression of an additional protein band reacting to P4HA1 specific antibody. (B) HFF cells and HEL cells also expressed a second protein reacting to antibody specific to P4HA1 at 6 hrs after VC (40 μM) treatment. (C) PNGaseF treatment of VC-treated and non-treated MEF cell lysate reduced the apparent molecular weight of the proteins reacting to antibody specific for P4HA1. (D) HA-tagged WT, N259Q, N113Q or N113Q/N259Q double mutants were expressed in P4ha1-silenced MEF cells in the absence of VC. The cell lysates were immunoblotted with HA-tag antibody. The N259Q mutant showed the same apparent molecular weight as WT, whereas the apparent molecular weights of N113Q and N113Q/N259Q double mutants were equal but lower than WT (left panel). WT, N113Q, N259Q and N113Q/N259Q double mutants all showed the same motility as N113Q or N113Q/N259Q double mutants after PNGase F treatment (right panel). (E) N259Q mutated P4HA1 did not show a motility shift after VC treatment (top panel) whereas PNGaseF treatment increased motility of N259Q mutated P4HA1 with or without VC stimulation (third panel). (F) 40 μM or higher concentrations of VC induced an additional protein band reacting to HA-tagged antibody for N113Q mutated P4HA1 (top panel), which was sensitive to PNGaseF treatment (third panel).