Figure 7. Analysis of Hrd1 expression and functions in human CD4 T cells.

(a) The levels of Hrd1 expression in freshly isolated CD4 T cells from healthy donors (N=12) or MS patients (N=16) by real-time PCR. (b–d) CD4 T cells freshly isolated CD4 T cells from healthy donors or MS patients (N=5) were lysed and subjected to western blotting analysis for the protein levels of Hrd1 and p27^kip1 using GAPDH as a loading control (b), and the relative band intensities were quantified (c). The mRNA levels of p27^kip1 in samples of (b) were determined by real-time PCR using β-actin as a control (d). (e–k) Purified CD4 T cells from healthy donors were infected with lentivirus carrying control or Hrd1-specific shRNA. (e,f), green fluorescent protein (GFP)-positive cells were sorted, the expression levels of Hrd1, p27^kip1 and GAPDH were examined by western blotting (e), and p27^kip1 ubiquitination was determined as in Fig. 4a (f). (g,h) Cells were stained with far-red cell trace and cultivated for 3 days with TCR/CD28 stimulation. GFP-positive cells were gated and the proliferation was analysed for CFSE dilution. (h,i) Cells were cultivated under Th1, Th17 and Treg polarization conditions and the gated GFP⁺ cells were analysed. Representative images (g,i,j) and the percentages from three independent experiments (h,k) are shown. Mann–Whitney test was used for the statistical analysis in a,c,d,h and k.