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. 2021 Aug 13;15:730417. doi: 10.3389/fnins.2021.730417

FIGURE 1.

FIGURE 1

Role of N-linked glycosylation in PKR2 trafficking. (A) Schematic depiction of human PKR2 glycosylation sites. (B) Western blot detection of untreated PKR2-GFP or PKR2-GFP treated with PNGase F or EndoH. (C,D) Western blot detection of PKR2 (C) or MRAP2 (D) following co-immunoprecipitation of PKR2-GFP and MRAP2-3XFLAG from transfected CHO cells. Immunoprecipitation was performed using beads coated with GFP nanobodies “G” or anti-Flag antibody “F.” “X” indicates that no antibody was used for the IP (beads only). (E) Western blot detection of PKR2-GFP alone or with MRAP2 treated or not with PNGase F or Endo H. (F–H) Densitometry analysis measuring the ration of glycosylated to non-glycosylated PKR2 in the presence and absence of MRAP2 for WT (F), N7Q mutant (G), and N27Q mutant (H). (I) Immunofluorescence detection of 2HA-PKR2, 2HA-PKR2-N7Q, 2HA-PKR2-N27Q, and 2HA-PKR2-N7/27 in non-permeabilized and permeabilized cells. (J,K) Cell ELISA detection of 2HA-tagged PKR2 and PKR2 mutants in non-permeabilized (J) and permeabilized (K) cells. Errors are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.