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. 2020 Dec 15;9:e63997. doi: 10.7554/eLife.63997

Figure 3. Validation of select reglucosylation substrates.

(A) The designated cell lines were lysed and split into whole cell lysate (WCL, 10%) or affinity purification by GST-CRT-WT or GST-CRT-Y109A and imaged by immunoblotting against the CI Man-6-Phosphate receptor. Data is representative of three independent experiments with quantification shown in panel (B). Quantifications were calculated by subtracting the value of protein in the Y109A lane from the value of protein in the associated wild-type (WT) lane, divided by the value of protein in the associated WCL lane and multiplied by 100. Error bars represent the standard deviation. Asterisks denote a p-value of less than 0.05. (C) Tandem mass tag (TMT) mass spectrometry quantification of CI Man-6-Phosphate receptor reglucosylation from ALG6/UGGT1-/- cells (Figure 2B) and ALG6/UGGT2-/- cells (Figure 2A). (D) Cartoon representation of CI Man-6-Phosphate receptor with N-glycans (branched structures), the signal sequence (gray), luminal/extracellular domain (blue), transmembrane domain (black), and intracellular domain (green) depicted. Number of amino acids and Cys residues are indicated. (E) Reglucosylation of IGF-1R, conducted as previously described above. Pro IGF-1R and mature IGF-1R are both observed due to proteolytic processing. Data are representative of three independent experiments with quantification displayed in (F). (G) TMT mass spectrometry quantification of IGF-1R from Figure 2A and B, as previously described. (H) Cartoon depiction of IGF-1R. (I) The reglucosylation of ENPP1 shown with quantification displayed in J. (K) TMT mass spectrometry quantification of ENPP1 from Figure 2A and B with cartoon depiction of ENPP1 in L. (M) Reglucosylation of β-hexosaminidase subunit β, conducted as previously described with quantifications displayed in N and TMT mass spectrometry quantification of β-hexosaminidase subunit β from Figure 2A and B in O with a cartoon depicting β-hexosaminidase subunit β in P.

Figure 3—source data 1. Quantifications for reglucosylation validations.

Figure 3.

Figure 3—figure supplement 1. mRNA expression of lysosomal preferential UDP-glucose:glycoprotein glucosyltransferase (UGGT)2 substrates.

Figure 3—figure supplement 1.

(A) The expression of HEXB (A), ARSA (B), NAGA (C), GLA (D), TPP1 (E), FUCA1 (F), HEXA (G), and NAGLU (H) in the indicated cell lines was analyzed by mRNA expression level in the denoted cell lines from RNAseq data presented in Supplementary file 4. The standard deviation from the average expression level in counts per million in each cell line for all genes is plotted. Error bars represent standard deviation. Data are representative of three independent experiments.
Figure 3—figure supplement 2. UPR induction in knockout cell lines.

Figure 3—figure supplement 2.

(A) The indicated cells were lysed and whole cell lysate were resolved by SDS-PAGE and imaged by immunoblotting against BiP and GAPDH. DMSO was used as a vehicle control with tunicamycin (5 μg/ml) as a positive control. Data is representative of three independent experiments with quantification displayed in (B). BiP expression levels were normalized to that of wild-type cells and the corresponding GAPDH loading control. Error bars denote standard deviation. Asterisks denote a p-value of less than 0.05. (C) A subset of genes induced by the ATF6 UPR branch was analyzed by mRNA expression level as described in Figure 3—figure supplement 1. Data are representative of three independent experiments. IRE1 (D) and PERK (E) induced genes characterized as described in (C).