Infrared dye coupled secondary antibodies

HIV-1ΔR8.2 (HIV-1NL4-3 R9ΔApa [70 (link)]) and HIV R9 were used. Its late domain mutants, ΔPTAP and ΔYP were previously described [43 ]. Humanized Gag was produced as previously described [71 (link)]. ALIX (NM_013374), Tsg101 (NM_006292), CHMP4b (NM_176812) and VPS4A (NM_013245) were kindly provided by Dr. Wesley Sundquist (university of Utah) and were all HA N-terminally tagged. GFP ORF was cloned from peGFP-N1 (Clontech). Point mutations were introduced using the Quick Change site directed mutagenesis kit (Stratagene).
All cell lines used were grown in complete DMEM medium under standard conditions, excepted for TIRF experiments where cells were incubated in CO2-independent medium (LifeTechnologies).
Anti-ALIX [44 (link)], anti-Tsg101 (C-2, Santa Cruz Biotech.), anti-HA (HA.11 clone 16B12, Covance), anti-p24 (183-H12-5C, NIH AIDS Reagent Program), anti-p17 (17–1, Santa Cruz Biotech.), anti-RT (MAb21, NIH AIDS Reagent Program), anti-PR (1696, Santa Cruz Biotech.), and infrared dye coupled secondary antibodies (LI-COR) were used for immunoprobing. Scanning was performed with the Odyssey infrared imaging system (LI-COR) in accordance with the manufacturer’s instructions at 700 or 800 nm, accordingly.

Samples were diluted in Laemmli buffer (10 mM Tris, pH 6.8, 1.5% SDS, 6% glycerol, 0.05% bromophenol blue, and 40 mM dithiothreitol) and subjected to SDS-PAGE. Immunoblot analysis using nitrocellulose membranes was performed as described previously48 (link). Both blocking buffer and infrared dye-coupled secondary antibodies were obtained from LI-COR (Lincoln, NE). Fluorescence signals from discrete bands were read out using the LI-COR Odyssey System. The rabbit polyclonal anti-AQP2 antibody used 1:2000 was described in Hoffert et al.48 (link). The phosphospecific antibody recognizing phosphorylated Ser5 present in heptad repeats in the COOH-terminal domain of Polr2a was purchased from Abcam (ab5131) and used at 1:1000 following the manufacturer’s protocol. Bands were quantified by densitometry.
Samples were diluted in Laemmli buffer and subjected to SDS-PAGE as previously described10 (link). Immunoblot analysis using nitrocellulose membranes was performed as previously described10 (link). The anti-AQP2 antibody (1:2000) was from Hoffert et al.48 (link). A rabbit polyclonal phosphospecific antibody recognizing phosphorylated Ser5 present in heptad repeats in the COOH-terminal domain of Polr2a was purchased from Abcam (ab5131) and used at 1:1000 following the manufacturer’s protocol. Bands were quantified by densitometry.