Staphylococcus aureus PGN and E coli LPS were purchased from Sigma-Fluka (St. Louis, MO, USA). Human CAECs from non-diabetic and diabetic donors (information in Table
1) were purchased from Lonza (Boulder, CO, USA). Protein assay reagents and ECL immunoblotting substrate were purchased from Pierce (Rockford, IL, USA). The following antibodies were used for Western-blot analysis: rabbit anti-human intercellular adhesion molecule (ICAM)-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal rabbit anti-human TLR2 (Imgenex, San Diego, CA, USA), monoclonal rabbit anti-human TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human phosphor-nuclear factor kappa B (NF-κB) p65 (Cell Signaling, Boston, MA), rabbit anti-human total NF-κB p65 (Cell Signaling, Boston, MA, USA), rabbit anti-human beta-actin (Cell Signaling, Boston, MA, USA), and rabbit anti-mouse ICAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). RNeasy micro kit was purchased from QiaGen (Valencia, CA, USA). IL-6 and IL-8 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).
Table 1
Details of the human CAECs used in this study
Non-diabetic | 3F0239 | 25y, ♂ | 3 | 4-6 |
Non-diabetic | 7F4019 | 56y, ♂ | 3 | 4-6 |
Non-diabetic | 7F4249 | 36y, ♂ | 3 | 4-6 |
T1D | 7F3422 | 56y, ♂ | 3 | 4-6 |
T1D | 7F4021 | 57y, ♂ | 3 | 4-6 |
T1D | 7F3795 | 34y, ♂ | 3 | 4-6 |