Hrp conjugated polyclonal rabbit anti mouse immunoglobulins

To detect the direct binding between non-core IFT-A complexes and lipids, the His-GFP-tagged IFT-A trimeric complex or His-tagged proteins purified from insect cells and Membrane Lipid Strips (Echelon Biosciences, P-6002) with 100 pmol of 15 different lipids were used following the manufacturer’s protocol. The strips were blocked in 3% (w/v) BSA in TBS-T buffer (50 mM Tris [pH 7.4], 150 mM NaCl, and 0.1% [v/v] Tween 20) at 4°C overnight in dark with gentle agitation. After blocking, they were washed in TBS-T buffer three times and 5 min each, followed by incubation at RT for 1 hr with IFT-A proteins in SEC buffer supplemented with 3% (w/v) BSA. The strips were washed three times in TBS-T as before and soaked in 3% (w/v) BSA in TBS-T with primary antibody against His-tag (THE His Tag Antibody, Mouse, GenScript) at a 1:2500 dilution for 1 hr at RT. Strips were washed three times and incubated with horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-mouse immunoglobulins (1:1000 dilution, Dako) for 1 hr followed by three TBS-T washes. An ECL Prime Western Blotting reagent (Amersham) was used as the substrate for the HRP, and the binding of IFT-A proteins onto spotted lipids was recorded with the ChemiDoc imaging system (Bio-Rad).

HIV-1 gp41-derived monomeric peptides were custom synthesized by Synpeptide Co Ltd (Shanghai, China) and the dimeric peptides by Pepscan Presto BV (Amsterdam, Netherlands). The antibody to gp160/41 (Chessie 8) and Vif (#319) were obtained through the NIH AIDS Research and Reference Reagent Program [41 (link)]. Polyclonal rabbit anti-Nef antibody was obtained from Thermo Fisher Scientific, Inc. Monoclonal mouse anti-β-actin antibody was obtained from Sigma. The antibody to p24 (EF7), has previously been described [42 (link)]. The secondary antibodies, HRP-conjugated polyclonal goat anti-rabbit immunoglobulins and HRP-conjugated polyclonal rabbit anti-mouse immunoglobulins, were obtained from DAKO.