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. 2019 Nov 13;9:16725. doi: 10.1038/s41598-019-53234-x

Figure 2.

Figure 2

DGC induces TXNIP down-regulation in T cells. (A) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from blood samples incubated for 4 hours (4 h blood, Procedure II, Fig. 1B) after no treatment (−) or centrifugation (+). (B) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells immediately isolated and lysed (0 h, Procedure I, Fig. 1B), isolated and incubated for 4 hours before lysis (4 h T cells, Procedure I, Fig. 1B), and isolated and lysed after incubation of PBMC for 4 hours (4 h PBMC, Procedure III, Fig. 1B) after DGC on either Lymphoprep, Histopaque or Ficoll-Paque as indicated. (C) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from blood samples incubated for 4 hours (4 h blood, Procedure II, Fig. 1B) with no addition (Control) or with Lymphoprep, Histopaque or Ficoll-Paque as indicated. (D) Overview of different T cell isolation and incubation procedures used. The text in the red frames gives the procedure used and the nomenclature for the T cell lysates analyzed and shown in (E) lanes 4–9. (E) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells immediately isolated and lysed (lane 1: 0 h, Procedure I, Fig. 1B; lane 4: monocyte depletion, 0 h, Procedure IV, (D); lane 7: T cell enrichment, 0 h, Procedure VI, D), isolated and incubated for 4 hours before lysis (lane 2: 4 h T cells, Procedure I, Fig. 1B; lane 5: monocyte depletion, 4 h T cells, Procedure IV, (D); lane 8: T cell enrichment, 4 h T cells, Procedure VI, D), and isolated and lysed after incubation of PBMC for 4 hours (lane 3: 4 h PBMC, Procedure III, Fig. 1B; lane 6: monocyte depletion, 4 h PBMC, Procedure V, D) or T cells for 5 h (lane 9: T cell enrichment, 5 h T cells, Procedure VI, D). (A–C,E) Each Western blot is representative for Western blots obtained from at least 3 different biological experiments and the quantification shows the mean + SEM of the band densities of TXNIP from Western blots obtained from at least 3 different biological experiments. The positions of the relevant molecular weight markers and their molecular weight in kDa are given to the right of each Western blot.