Fig. 5: RNF128 reduces TLR4 protein level.
From: RNF128 regulates neutrophil infiltration and myeloperoxidase functions to prevent acute lung injury
A Immunoblotting analysis of TLR4 expression in the lung samples from WT and RNF128 KO mice with or without LPS treatment for 24 h. Relative immunoblotting signals were inducted. B, C Endogenous TLR4 levels in the indicated stable cell lines with or without LPS treatment. D TLR4 degradation in cells overexpressing Ring mutant RNF128 or WT cells. HEK293 cells were transfected with 0.5 μg of vector, RNF128, or RNF128 (H2N2) in the presence of 0.5 μg of TLR4. The cells were subsequently harvested and subjected to immunoblotting. E HEK293 cells were transfected with 0.5 μg of vector, RNF128, or TLR4 and then treated with Mg132 (50 μM) for 8 h. The cells were subsequently harvested and subjected to immunoblotting and the data quantified using Image J software. (F) HEK293 cells were transfected with 0.5 μg of vector, RNF128 or TLR4 and then treated with NH4CL (50 mM) for 8 h. The cells were subsequently harvested and subjected to immunoblotting and the data were quantified using Image J software. G, H MH-S/vector and MH-S/RNF128 cells were treated with cycloheximide (CHX, 100 μM) and harvested at the indicated times. Cell lysates were subjected to immunoblotting using the indicated antibodies. The data are presented as mean ± SD. The statistical significance was evaluated using the Student’s t-test. ***P < 0.001.
