Fig. 4

CREB phosphorylation by Tat and its requirement for Tat basic domain. a, b U373.MG were transfected with Tat.His or basic domain-deleted Tat (ΔBD.His) plasmid, cultured for 48 h and harvested for cell lysates, followed by Western blotting (a), or RNA isolation, followed by qRT-PCR for miR-132 level (b). C3 was used as the control, and snRNA U6 was used to normalize miR-132 level. A control miRNA was included to equalize the input miRNA. The qPCR data were mean ± SD of triplicates and representative of three independent experiments. c Primary human astrocytes were treated with recombinant Tat protein at 0, 5, 10, 100, and 200 ng/ml for 24 h. Cell lysates were then prepared for Western Blotting for CREB and pCREB. β-actin was included as a loading control for Western blotting. Western blots were quantitated using ImageJ software. The relative protein level was calculated using β-actin as the reference, and the first control sample was set at 1.0. The data were representative of three independent experiments