Anti mll1c

Antibodies used included anti-Flag (Sigma, F3165-5MG, clone M2), anti-MLL1C (Bethyl, A300-374A), anti-RbBP5 (Bethyl, A300-109A), anti-ASH2L (Bethyl, A300-112A), anti-WDR5 (Bethyl, A302-430A), anti-GFP (Clontech, 632377, clone JL-8), anti-trimethyl-Histone H3 (Lys4) (EMD Millipore, 07-473), anti-LANA LN53 (Millipore, MABE1109), goat anti-rat IgG (H + L) Alexa Fluor 488 (Invitrogen A11006), goat anti-rabbit IgG (H + L) Alexa Fluor 647 (Invitrogen A21245), human anti -LANA sera adsorbed against uninfected cell extract for western blot or affinity purified against carboxy-terminal LANA for ChIP, Anti-T7 tag^® antibody (Abcam, ab9138), and anti-alpha-tubulin (Sigma T9026, cline DM1A).

1 × 10⁵ BCBL1 cells were incubated in a well of a 12-well slide chamber (Ibidi) in 200 μl RPMI with 10% BGS for 24 h at 37°C. Supernatant was removed and cells fixed with 4% paraformaldehyde in PBS for 15 min. After fixation, cells were rinsed ×2 with 1× PBS, and blocked/permeabilized with block/perm buffer (10% BGS, 100 mM glycine, 0.2% Triton-X100 in PBS) with the addition of 5 μg/sample of human IgG. After 30 min, the block/perm buffer was removed and cells were incubated with primary antibodies anti-LANA LN53 (Millipore) and anti-MLL1C (Bethyl), or anti-LANA LN53 and anti-WDR5 (Bethyl) diluted in block/perm buffer at 1:200. After 1h of incubation at room temperature, antibodies were removed, and cells washed ×2 with 1× PBS. Secondary antibodies goat anti rat Alexa Fluor 488 (Invitrogen) and goat anti rabbit Alexa Fluor 647 (Invitrogen)) were added at 1:1000 dilutions, and incubated for 1h at room temperature in dark. Cells were washed ×2 with 1× PBS and coverslips mounted using ProlongGold antifade with DAPI (Life Technologies). Images were captured using a Zeiss LSM 800 confocal microscope and 63× objective. Co-localization was quantified using Zen Blue software and Pearson's correlation coefficient.