mouse monoclonal anti gp96  (R&D Systems)

Journal: The Journal of Cell Biology

Article Title: Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites

doi: 10.1083/jcb.201904004

Figure Lengend Snippet: Hyperglycosylation of GRP94. (A) The domain structure and location of glycosylation sites in GRP94. (B) The Δlog 2 values for GRP94 sites in STT3A −/− and STT3B −/− cells. Error bars designate SDs ( n = 3–8) unless denoted by an asterisk ( n = 2). Not quantifiable (NQ) and not detected (ND) report results for the STT3B −/− cells. (C) Protein (50 µg) in lysates from WT and mutant cells was analyzed by protein immunoblotting using antisera to GRP94 and the α-subunit of ATP synthase. EH designates digestion with EH. (D) EH digestion time course of pulse-labeled GRP94. (E) Pulse labeling of GRP94 in similar numbers of WT and mutant cells. The right hand lane is a lighter version of the preceding lane. (F) Pulse labeling (5 min) of GRP94 in STT3A −/− cells followed by the indicated chase times. (G) Pulse labeling of human GRP94-DDK-His and GRP94-DDK-His N217Q mutant in WT and mutant cells. (H) Pulse labeling of GRP94 in WT or STT3A −/− cells treated with the following compounds: DTT (200 µm), tunicamycin (Tn, 0.6 µM), thapsigargin (Tg, 0.1 µM), and NGI-1 (10 µM). (I) Pulse labeling of GRP94 in control fibroblasts (C-1 and C-2), STT3A-CDG fibroblasts, and WT and STT3A −/− cells. The EH-digested sample is from the STT3A −/− cells and is equivalent to 10% of the undigested sample.

Article Snippet: Mouse monoclonal antibodies to GRP94 (MAB7606; R&D Systems), GLUT1 (ab40084; Abcam), the α-subunit of the F o F 1 ATPase (612516; BD Biosciences), and the DDK epitope tag (F3165 anti-FLAG M2; Sigma-Aldrich) were obtained from commercial sources.

Techniques: Glycoproteomics, Mutagenesis, Western Blot, Labeling, Control