Figure 2.
Figure 2. N-Hyperglycosyation of SAMD14 and neurabin-I in PCNSL. (A) N-hyperglycosylation of SAMD14 and neurabin-I in patients with SAMD14/neurabin-I-reactive lymphoma BCRs. Deglycosylation of SAMD14 and neurabin-I from PCNSL tissue with a panel of endo- or exoglycosidases (PNGase-F, O-glycosidase, neuraminidase, β-n-acetylglycosidase and galactosidase). (Top) Lane 1: untreated PCNSL tissue from patient #3 (without SAMD14 reactive lymphoma BCR). Lane 2: PNGase-F treated PCNSL tissue from patient #3. Lane 3: untreated PCNSL tissue from patient #6 (with SAMD14-reactive lymphoma BCR). Lane 4: PNGase-F-treated PCNSL tissue from patient #6. Lane 5: O-glycosidase-treated PCNSL tissue from patient #3. Lane 6: O-glycosidase-treated PCNSL tissue from patient #6. Lane 7: α-2(3,6,8,9)-neuraminidase-treated PCNSL tissue from patient #3. Lane 8: α-2(3,6,8,9)-neuraminidase treated PCNSL tissue from patient #6. (Bottom) Lane 1: untreated PCNSL tissue from patient #3. Lane 2: PNGase-F-treated PCNSL tissue from patient #3. Lane 3: untreated PCNSL tissue from patient #6. Lane 4: PNGase-F-treated PCNSL tissue from patient #6. Lane 5: β-N-acetylglucosaminidase-treated PCNSL tissue from patient #3. Lane 6: β-N-acetylglucosaminidase-treated PCNSL tissue from patient #6. Lane 7: β-1→4-galactosidase-treated PCNSL tissue from patient #3. Lane 8: β-1→4-galactosidase-treated PCNSL tissue from patient #6. The treatment with PNGase-F and β-N-acetylglucosaminidase resulted in the disappearance of the differences in molecular weights of both antigens from patients #3 and #6. (B) Deglycosylation of SAMD14 and neurabin-1 from PCNSL tissue only with β-N-acetylglucosaminidase. The treatment with β-N-acetylglucosaminidase resulted in the disappearance of the differences in molecular weights of both SAMD14 and neurabin-I from patients #3 and #6. (C) Identification of the hyperglycosylated Asn residues. Western blots of several constructs of SAMD14 and neurabin-I with different mutants of N-glycosylation sites expressed in LCLs derived from patients with and without hyperglycosylated SAMD14 and neurabin-I. Anti-FLAG antibodies were used as primary antibodies. These western blots showed the disappearance of the gain in molecular weight for the point-mutated, full-length proteins N339Q SAMD14 and N1277Q neurabin-I expressed in LCLs of patients with PCNSL with SAMD14/neurabin-I antibodies. (D) Identification of the hyperglycosylated Asn residues. Western blots of several constructs of SAMD14 and neurabin-I with different mutants of N-glycosylation sites expressed in LCLs derived from patients with and without hyperglycosylated SAMD14 and neurabin-I. Instead, Anti-FLAG primary antibody recombinant SAMD14/neurabin-I-specific Fab was used as primary antibody. These western blots show double bands as they unspecifically stain for both wild-type SAMD14 (hyperglycosylated) and nonhyperglycosylated N339Q SAMD14, and for both wild-type neurabin-I (hyperglycosylated) and N1277Q neurabin-I.

N-Hyperglycosyation of SAMD14 and neurabin-I in PCNSL. (A) N-hyperglycosylation of SAMD14 and neurabin-I in patients with SAMD14/neurabin-I-reactive lymphoma BCRs. Deglycosylation of SAMD14 and neurabin-I from PCNSL tissue with a panel of endo- or exoglycosidases (PNGase-F, O-glycosidase, neuraminidase, β-n-acetylglycosidase and galactosidase). (Top) Lane 1: untreated PCNSL tissue from patient #3 (without SAMD14 reactive lymphoma BCR). Lane 2: PNGase-F treated PCNSL tissue from patient #3. Lane 3: untreated PCNSL tissue from patient #6 (with SAMD14-reactive lymphoma BCR). Lane 4: PNGase-F-treated PCNSL tissue from patient #6. Lane 5: O-glycosidase-treated PCNSL tissue from patient #3. Lane 6: O-glycosidase-treated PCNSL tissue from patient #6. Lane 7: α-2(3,6,8,9)-neuraminidase-treated PCNSL tissue from patient #3. Lane 8: α-2(3,6,8,9)-neuraminidase treated PCNSL tissue from patient #6. (Bottom) Lane 1: untreated PCNSL tissue from patient #3. Lane 2: PNGase-F-treated PCNSL tissue from patient #3. Lane 3: untreated PCNSL tissue from patient #6. Lane 4: PNGase-F-treated PCNSL tissue from patient #6. Lane 5: β-N-acetylglucosaminidase-treated PCNSL tissue from patient #3. Lane 6: β-N-acetylglucosaminidase-treated PCNSL tissue from patient #6. Lane 7: β-1→4-galactosidase-treated PCNSL tissue from patient #3. Lane 8: β-1→4-galactosidase-treated PCNSL tissue from patient #6. The treatment with PNGase-F and β-N-acetylglucosaminidase resulted in the disappearance of the differences in molecular weights of both antigens from patients #3 and #6. (B) Deglycosylation of SAMD14 and neurabin-1 from PCNSL tissue only with β-N-acetylglucosaminidase. The treatment with β-N-acetylglucosaminidase resulted in the disappearance of the differences in molecular weights of both SAMD14 and neurabin-I from patients #3 and #6. (C) Identification of the hyperglycosylated Asn residues. Western blots of several constructs of SAMD14 and neurabin-I with different mutants of N-glycosylation sites expressed in LCLs derived from patients with and without hyperglycosylated SAMD14 and neurabin-I. Anti-FLAG antibodies were used as primary antibodies. These western blots showed the disappearance of the gain in molecular weight for the point-mutated, full-length proteins N339Q SAMD14 and N1277Q neurabin-I expressed in LCLs of patients with PCNSL with SAMD14/neurabin-I antibodies. (D) Identification of the hyperglycosylated Asn residues. Western blots of several constructs of SAMD14 and neurabin-I with different mutants of N-glycosylation sites expressed in LCLs derived from patients with and without hyperglycosylated SAMD14 and neurabin-I. Instead, Anti-FLAG primary antibody recombinant SAMD14/neurabin-I-specific Fab was used as primary antibody. These western blots show double bands as they unspecifically stain for both wild-type SAMD14 (hyperglycosylated) and nonhyperglycosylated N339Q SAMD14, and for both wild-type neurabin-I (hyperglycosylated) and N1277Q neurabin-I.

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