Nuclear Factor-Kappa B Family Member RelB Inhibits Human Immunodeficiency Virus-1 Tat-Induced Tumor Necrosis Factor-Alpha Production
Figure 4
Tat induces de novo synthesis of RelB.
A, BV-2 cells (1.2×105) were treated with Tat (100nM) for the indicated periods of time and whole cell lysates were subjected to immunoblot analysis using either RelB-specific (upper panel) or α-Tubulin-specific (lower panel) antibodies. B, BV-2 cells (1.2×105) were treated with Tat alone or together with CHX as indicated for 4h. Whole cell lysates were subjected to immunoblot analysis using either RelB-specific (upper panel) or α-Tubulin-specific (lower panel) antibodies. C, BV-2 cells (1.2×105) were treated with Tat (100nM) alone or together with MG-132 or TPCK both at a concentration of 50µM for the indicated periods of time. Whole cell lysates were subjected to immunoblot analysis using a RelB-specific (upper panel) antibody. Levels of a nonspecific (n.s.) band (lower panel) are shown to indicate equal protein loading. D, BV-2 cells (1.2×105) were treated with gp120 (SF162, 100nM) for the indicated periods of time and whole cell lysates were subjected to immunoblot analysis using either RelB-specific (upper panel) or α-Tubulin-specific (lower panel) antibodies. E, Primary human monocytes (2×105) were treated with Tat for the indicated periods of time and whole cell lysates were subjected to immunoblot analysis using either RelB-specific (upper panel) or Actin-specific (lower panel) antibodies. F, Primary human monocytes (2×105) were treated with Tat for the indicated periods of time and whole cell lysates were subjected to immunoblot analysis using either p100/p52-specific (upper panel) or Actin-specific (lower panel) antibodies. G, BV-2 cells (1.2×105) were treated with Tat for the indicated periods of time and whole cell lysates were subjected to immunoblot analysis using either p100/p52-specific (upper panel) or α-Tubulin-specific (lower panel) antibodies. H, BV-2 cells (1.2×105) were treated with Tat as indicated and cytosolic (labeled “C”) and nuclear (labeled “N”) extracts were subjected to immunoblot analysis with either RelB-specific (upper panel) or α-Tubulin-specific (lower panel) antibodies. I, Primary human astrocytes (4×104) were plated 72h prior to treatment. These cells were treated with Tat as indicated and whole cell lysates were subjected to immunoblot analysis using either RelB-specific (upper panel) or α-Tubulin-specific (lower panel) antibodies.