Anti erp57 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).

The cells were initially plated in triplicate at a density of 2.5 × 10⁴ cells/well in 96 well plates. For the treated samples, hIAPP was added to the cells to final concentrations of 5 μM per well. Plates were incubated for 24 h at 37 °C. The In Cell Western assay was then carried out using In-Cell Western™ Kit II (LI-COR Biosciences, UK) according to the manufacturer’s instructions. Primary antibodies for this assay were used as follows: Anti-PCNA antibody, 1:100; Anti-EEF2 antibody, 1:120; Anti-Superoxide Dismutase 1 antibody, 1:200; Anti-Peroxiredoxin1 antibody, 1:250; Anti-ERp57 antibody,1:500; Anti-14-3-3 zeta antibody 1:500 (all Abcam-UK). Donkey anti-rabbit-IRDye 700CW (LI-COR Biosciences, UK) secondary antibody was diluted 500-fold and used for the detection of the primary antibodies. The Odyssey Infrared Imaging System (LI-COR Biosciences, UK) was then used to scan the plate and to detect the signal from the secondary antibody in the 800 nm channel.