human embryonal carcinoma  (ATCC)

Journal: Glycobiology

Article Title: Identification of C -mannosylation in a receptor tyrosine kinase AXL

doi: 10.1093/glycob/cwae096

Figure Lengend Snippet: DPY19 family proteins are not C -mannosyltransferases of AXL. (A) Establishment of AXL-ECD and DPY19 family-co-expressing S2 cells. S2 cells were transfected with pMT-PURO-AXL-ECD and pIZ-DPY19L1, L2, L3, or L4. mRNA expression levels of DPY19 family were measured by RT-PCR. Mock S2 cells were co-transfected with pMT-PURO-AXL-ECD and pIZ/V5-his empty vector. (B) C -mannosylation of AXL is not catalyzed by DPY19 family proteins. Recombinant AXL-ECD was purified from conditioned media of DPY19 family-expressing S2 cells. These samples were analyzed by LC–MS/MS. Only unmodified 309 VACTSSQGPSSWTHWLPVETPE 330 peptides were detected in samples from mock and DPY19L1-, L2-, L3-, and L4-expressing cells as m / z = 1235.6. (C and D) mRNA expression level of DPY19 family in MDA-MB-231 cells. cDNA of HT1080 and NEC8 cells were used as a positive control of DPY19L1, L3, and L4 (HT1080, C), and DPY19L2 (NEC8, D).

Article Snippet: Human NSCLC PC9 (American Type Culture Collection, Manassas, VA) and H3122 cells (gifted by Dr. JA Engelman, Massachusetts General Hospital Cancer Center, Boston, MS), and human embryonal carcinoma NEC8 cells (RIKEN BioResource Center) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% fetal bovine serum, 100 mg/L kanamycin, 100 units/mL penicillin G, 300 mg/L L-glutamine, and 2.25 g/L NaHCO 3 , at 37 °C with 5% CO 2 .

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Recombinant, Purification, Liquid Chromatography with Mass Spectroscopy, Positive Control