Secinh3

The phospho-(Ser) PKC substrate (pSer PKC substrate) antibody was from Cell Signaling Technology (Danvers, MA, USA). The cytohesin-2 western blot antibody, ARF6 antibody and GAPDH antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The cytohesin-2 immunoprecipitation antibody was from Thermo Scientific (Loughborough, Leicestershire, UK). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit, anti-mouse and anti-goat secondary antibodies were from Jackson ImmunoResearch Laboratories (Newmarket, UK). The PKC inhibitor bisindolylmaleimide (BIM) IX (Ro 31-8220) and the inactive analogue BIM V were from Calbiochem (Nottingham, UK). The PKC inhibitors BIM I (GF 109203X), Go 6983 and ruboxistaurin (LY 333531) and the cytohesin-2 inhibitor SecinH3 were from Tocris (Bristol, UK). The GST-GGA3 construct was a generous gift from Professor Sidney Whiteheart (University of Kentucky, USA). Cross-linked collagen-related peptide (CRP) was synthesized by Professor Richard Farndale (University of Cambridge, UK). PE-P-selectin and FITC-PAC1 antibodies were from Emfret Analytics (Eibelstadt, Germany). Luciferin-luciferase was from Chronolog (Labmedics, Stockport, UK). NuPAGE LDS sample buffer was from Invitrogen (Carlsbad, CA, USA). ECL reagent was from GE Healthcare (Amersham, UK). All other reagents were from Sigma-Aldrich (Poole, UK).

The rat Müller cells (rMC-1) were obtained as a gift from Vijay Sarthy, Northwestern University, Chicago, Illinois, United States. Cells were cultured in DMEM media that was supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% of antibiotic-antimycotic (Thermo Fisher Scientific). The rMC-1 cells were authenticated using short tandem repeat analysis, contamination for interspecies and mycoplasma (IDEXX Bioresearch, Columbia, MO, USA). For insulin study, cells were serum starved in DMEM for 18 hours and then stimulated with 10 nM insulin (Sigma-Aldrich Corp., St. Louis, MO, USA) in the presence or absence of the inhibitor, SecinH3 (Tocris Bioscience, Bristol, UK). DMSO-treated cells (0.2% final concentration of DMSO) were used as a control. Cells were collected, washed with PBS and stored at −80°C.