Monoclonal mouse anti gfp

Infected primary cortical neurons were harvested with RIPA lysis buffer (50 mM Tris–HCl, pH 7.6; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS) containing protease inhibitor cocktail (Complete, Roche) 7 days after rAAV infection. Brain tissue (cortex and hippocampus) was collected 8 weeks after virus injection and homogenized in 25 mM Hepes buffer containing a protease inhibitor cocktail on ice. Proteins were separated by SDS-PAGE (10% separating and 4% stacking gels) and transferred onto nitrocellulose membranes. Immunoblots were probed with monoclonal mouse anti-GFP (1:8000, Clontech), polyclonal rabbit anti-2A (1:2000, Millipore), polyclonal rabbit anti-phospho-Smad1/5/8 (1:1000, Cell Signaling Technology), polyclonal rabbit anti-BMP7 (1:1000, Abcam), and monoclonal mouse anti-β-actin (1:5000) antibodies. Horseradish peroxidase-linked anti-rabbit or anti-mouse was used as secondary antibodies (1:15,000, Vector Laboratories), and blots were visualized by enhanced chemiluminescence (ECL kit, GE Healthcare). Immunoblots were quantified using ImageJ (NIH).

Cell lysates were prepared by resuspending washed cell pellets directly in 2.5 X Laemmli sample buffer. Viral particles were purified from the filtered supernatant by centrifugation prior to resuspension in 2.5X Laemmli sample buffer. A3-HA was detected with monoclonal mouse anti-HA (BioLegend), Vif-MYC was detected with polyclonal rabbit anti-MYC (Sigma-Aldrich), Tubulin (TUB) was detected with monoclonal mouse anti-α-Tubulin (Covance), HIV-1 Gag was detected with monoclonal mouse anti-HIV-1 p24 (NIH AIDS Reagent Program) [54 (link)], A3-GFP was detected with monoclonal mouse anti-GFP (Clontech), HSP90 was detected with mouse anti-HSP90 (BD Biosciences). A3B was detected with rabbit monoclonal anti-A3B [55 ] (Brown et al., in prep). To determine endogenous huA3B degradation, the huA3B and Tubulin bands were quantified from immunoblots using ImageJ (1.42q), and huA3B levels were normalized to those of Tubulin. These values were analyzed using a two-way ANOVA. Bonferroni's method for post-hoc testing was used to compare the amount of huA3B in the presence of vector, HIV-1_IIIB Vif, and SIVmac239 Vif. Statistical analyses were done with Prism 5 (GraphPad Software Inc.).

Proteins were separated by SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane by wet blot. Primary antibodies used for detection were: anti-CSN5 mouse monoclonal Santa Cruz Biotechnology sc-393725, anti-Cul1 mouse monoclonal Santa Cruz Biotechnology sc-17775, anti-Cul2 rabbit polyclonal Thermo Scientific #51–1800, anti-Cul3 rabbit polyclonal Cell Signaling #2769, anti-Cul4A rabbit polyclonal Cell Signaling #2699, anti-Cul5 rabbit polyclonal Bethyl Laboratories A302-173A, anti-β-actin mouse monoclonal Sigma A5316, anti-GFP mouse monoclonal Clontech #632381.