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. 2021 May 25;10(6):1310. doi: 10.3390/cells10061310

Figure 2.

Figure 2

RPS3 is fucosylated in vitro and in vivo. (A) RPS3 is recognized by UEAI lectin by agarose-bound lectin pull-down (PD). Cell lines IPC298, MelJuso, and SK-MEL-119 were cultured in growth media and harvested for protein lysate. Lysates were incubated with bead (agarose bead control), AAL (AAL-agarose), or UEAI (UEAI-agarose) to bind fucosylated proteins. UEAI-recognized RPS3 in all three cell lines. (B) Liver, muscle, brain, and skin samples were harvested from a C57BL/6 mouse and lysed using a Dounce homogenizer. Lysates were subjected to UEAI PD to isolate UEAI-recognized proteins. RPS3 was recognized by UEAI in all tissue types. LE: long exposure. (C) Cells transduced and selected with lentivirus containing Cas9/no guide (ng) or Cas9/sgSLC35C1 (gC1) were grown in normal growth media and harvested for protein lysate (Figure 1B). Lysate were subjected to UEAI PD to isolate UEAI-recognized proteins. Knockout of the Golgi fucose transporter SLC35C1 did not affect the ability of UEAI to recognize RPS3. (D) Cells expressing myc-DDK (FLAG)-tagged RPS3 (RPS3-mDDK) were harvested for FLAG IP and Western blot. The membrane was probed with mouse anti-FLAG/anti-mouse-680 and UEAI-biotin/strep-800 to assess direct recognition of RPS3 by UEAI (signal overlap by LI-COR imaging). UEAI recognizes RPS3. (E) Cells were grown in normal growth media and harvested for UEAI PD. The lectin PD was supplemented with either no sugar (control), L-fucose (Fuc), glucose (Glc), rhamnose (Rham), or galactose (Gal) at 1 M final concentration. Analysis by Western blot showed that fucose and rhamnose interfere with UEAI lectin recognition of RPS3, whereas glucose and galactose did not affect lectin binding. (F) Cells grown in the presence of labeled fucose were analyzed to assess labeling of RPS3. Labeled lysates or unlabeled lysates (control) were subjected to neutravidin pull-down (Neu-PD) to isolate proteins that integrated the labeled fucose. RPS3 did integrate the labeled fucose. β-actin, a non-fucosylated protein, did not show any integration of the labeled fucose (negative control).