Figure 4

BAG6 depletion stimulates the accumulation of mislocalized HLA-A protein in the membrane-associated fraction. (a) HeLa cells expressing N-terminally T7-tagged HLA-A*6802 were treated with (+) or without (−) MG-132 for 4 h, and were fractionated into cytosolic and membrane-associated/insoluble fractions. Both T7- and Flag-immunosignals were detected. Tubulin was used as a cytoplasmic marker and calnexin was used for the ER membrane fraction. (b) HeLa cells with an N-terminally T7-tagged HLA-A*6802 expression vector (or a mock vector) were treated with (+) or without (−) 10 μM MG-132, and harvested with PBS containing 1% Triton X-100 detergent. The detergent-insoluble protein fractions were subjected to anti-T7 western blot analysis. The successful isolation of microsome-free protein aggregates was verified by anti-Bip (negative), anti-Vimentin (positive) and anti-polyubiquitin FK2 (positive) immunoblots. (c) HeLa cells transfected with 5 nM bag6 siRNA#1 (+) or control siRNA (−) were fractionated into cytosolic and insoluble fractions, and were probed with anti-T7-antibodies to detect the MLP form of HLA-A*6802. The BAG6 blot indicates the depletion of BAG6 protein by its siRNA. (d) HeLa cells were transfected with an expression vector encoding N-terminally T7-tagged HLA-A*6802 protein with a Flag-tag at downstream of the SS (HLA-transfection). Empty vector transfection was used as a negative control (Mock transfection). The cells were treated with 10 μM MG-132 (for 4 h) or bag6 siRNA (for 72 h) as indicated. Microsome fractions were prepared from these cells, and they were probed with anti-T7 and anti-Flag antibodies. The microsome fraction contained Flag-positive HLA signals irrespective of the presence or absence of MG-132, while the T7-positive HLA signal was absent in this membrane fraction without MG-132. Note that bag6 siRNA greatly stimulated the accumulation of T7-positive MLP-HLA in the microsomal fraction. (e) HeLa cells were transfected with bag6 siRNA#1 duplex or control siRNA (5 nM each), and the lysates were probed with anti-T7- or anti-Flag-antibodies to detect MLP or total HLA-A*6802 species in non-fractionated cell extracts, respectively. The BAG6 blot indicates the near complete depletion of BAG6 protein by its siRNA. Actin was used as a loading control. Asterisks indicate non-specific signals. Full-length images of blots are presented in Supplementary Figs S4 and S5.