Figure 3
From: Effect of SWI/SNF chromatin remodeling complex on HIV-1 Tat activated transcription
Identification of cellular proteins associated with the transcriptionally active nucleosomal HIV-1 promoter. A) Nucleosomes were assembled on biotin labeled HIV-1 LTR DNA and G1/S extracts were added to the reaction mixture. Following washes (TNE600 plus 1% NP-40), samples were eluted with excess biotin and centrifuged to pellet the beads. Supernatants were TCA precipitated and ran on a 4–20% gel and silver stained (MALDI compatible). Lane 1, Strepavidin beads; lane 2, purified core histones before nucleosomal assembly; lane 3, purified Tat; lanes 4–7 are LTR sequences (-110/+180) from four different clade B isolates assembled and incubated with G1/S extracts without any nucleotide addition; lane 8, transcriptionally active HXB2 promoter with nucleotide addition (ATP, CTP, GTP); lane 9, HXB2 promoter with purified Tat; lane 10 same as lane 8 with purified Tat; lane 11, the rainbow molecular marker (14–220 kDa). B) Control gel utilizing HXB2 wild-type, TATA-/TAR+, and TATA+/TAR- promoters with Tat and nucleotide (ATP, CTP, GTP) and AdML promoters. Unique proteins (.) bound to DNA were cut out, trypsin digested, and used for identification by mass spectrometry [74].