Anti pdi antibody

Ribostamycin, PDI, BfA, GM1, GSH, CTA, S-nitrosoglutathione, and anti-CTB antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Bacitracin was purchased from Calbiochem (La Jolla, CA), CT was from List Biological Laboratories (Campbell, CA), and phosphate-buffered saline (pH 7.4) with 0.05% Tween 20 (PBST) was from Medicago (Uppsala, Sweden). ERp57, the anti-ERp57 antibody, and the anti-ERp72 antibody were from Abcam (Cambridge, MA). ERp72 and the anti-PDI antibody were purchased from Enzo Life Sciences (Farmingdale, NY). The anti-CTA1 monoclonal antibody 35C2 [52] (link) was a generous gift from Dr. Randall K. Holmes (University of Colorado School of Medicine). The pOLR130 plasmid encoding mature human PDI with an N-terminal His₆ tag [53] (link) was generously provided by Dr. Lloyd Ruddock (University of Oulu, Finland). Uniformly ¹³C-labeled ¹³C₆-_D-glucose and D₂O were purchased from Cambridge Isotope Laboratories (Andover, MA). Uniformly ¹³C-labeled CTA1-His₆ was produced as described in [7] (link) and purified as described in [22] (link).

DF1 cells seeded onto glass coverslips were transfected with pEGFP-N1-M41 nsp3, pmCherry-N1-BeauR nsp4, pmCherry-N1-M41 nsp4, pcDNA3.1(-)-BeauR nsp6-3xFLAG, and pcDNA3.1(-)-M41 nsp6-3xFLAG alone or in combination using lipofectamine 2000. Cells were transfected with a total of 500 ng plasmid with a DNA:lipofectamine 2000 ratio of 1:2 following the manufacturer’s instructions. After 24 h, cells were fixed for 20 min in 4% paraformaldehyde in PBS at room temperature. Cells were then permeabilized in 0.1% Triton X-100 in PBS for 10 min and blocked in 0.5% BSA in PBS for 1 h. Primary anti-FLAG M2 antibody (Sigma Aldrich) and anti-PDI antibody (Enzo Life Sciences, Exeter, UK) were diluted in blocking buffer and cells incubated for 1 h. After three washes in PBS, Alexa fluor conjugated secondary antibodies (Fisher Scientific) were diluted 1/500 and cells incubated for 1 h. After a further three washes in PBS, nuclei were strained using ToPro3 (Fisher Scientific) or DAPI (Sigma Aldrich) and coverslips mounted with Vectashield (Vector Laboratories, Peterborough, UK). Cells were visualized using a Leica SP5 confocal microscope (Leica Microsystems, Milton Keynes, UK). Quantitation of transfected cells was performed manually on three randomly selected fields of view.