Hrp conjugated anti mouse igg

Western blotting was performed as described in our previous studies [51 (link),56 (link),59 (link)]. Briefly, purified synaptosomes (7.5 µg) from each animal from the two groups were loaded onto 10% Bis-Tris wells (Invitrogen, Waltham, MA, USA) under reducing conditions, followed by transfer to a nitrocellulose membrane using iBlot2 (Invitrogen, Waltham, MA, USA) and immunodetection. Nonspecific antibody blocking was performed using Superblock (ThermoFisher, Waltham, MA, USA). Immunoblotting was performed with primary antibodies overnight at 4 °C against ADD1 (1:1000, ProteinTech, Rosemont, IL, USA) and GAPDH (1:2500, Invitrogen, Waltham, MA, USA), followed by secondary antibody (1:2500, HRP-conjugated anti-rabbit IgG; Thermo Scientific, Waltham, MA, USA) and (1:2500, HRP-conjugated anti-mouse IgG; Thermo Scientific, Waltham, MA, USA) against ADD1 and GAPDH, respectively. Primary and secondary antibody dilutions were carried out according to the manufacturer’s suggestions. Blots were developed using Azure CSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

Anti-actin monoclonal antibody (C4) and anti-LC-3 antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and Medical & Biological Laboratories (MBL; Nagoya, Japan), respectively. As the secondary antibodies, we used horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Thermo Fisher Scientific) for anti-actin monoclonal antibody (C4), and HRP-conjugated anti-rabbit IgG (Thermo Fisher Scientific) for anti-LC3 antibody. In Western blotting, the primary antibodies were diluted 1000-fold and the secondary antibodies were diluted 3000-fold with Tris-buffered saline with 0.05% Tween 20 (TBS-T).

The active form of Arf6 was detected using a GTP-bound Arf6-specific pulldown assay kit (Cell biolabs, STA-407-6). Cells were lysed in Arf6 lysis buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 0.2 mM EDTA and 2% glycerol, followed by centrifugation at 15,000 rpm for 10 min at 4°C. The supernatant was incubated with Golgi-localized γ-ear containing Arf-binding protein 3 protein binding domain (GGA3 PBD) agarose beads for 1 hr at 4°C. Beads were washed 3 times in Arf lysis buffer. The pellets were added to 2x Laemmli sample buffer and boiled at 95°C for 5 min. The supernatants were analyzed with SDS-PAGE and western blot analysis with Arf6 antibody (Cell Biolabs, STA-407-6, 1:500). HRP-conjugated anti-mouse IgG (Thermo Fisher Scientific, 32430) was used as the secondary antibody at 1:1000 dilution.

ELISA’s were performed following standard procedure. 2HB plates (96-well; Nalgene Nunc, Rochester, NY) were coated overnight with 90 ng/well HIV-1 Env-CN54 protein, diluted in 1X coating buffer (Cat # 6247ImmunoChemistry Technologies, Bloomington, MN), then washed with buffer (150 mM NaCl, 0.1% Tween-20), blocked for 1 hour in 5% nonfat milk, 3% heat-inactivated goat serum and 0.2% Tween-20 in PBS and then washed again. Serum samples were serially diluted in blocking buffer, and then added to the wells in duplicate and incubated for 2 hours at 37 °C. The plates were then washed and HRP-conjugated anti-mouse IgG (Thermo Scientific) was added at 1:2500 dilution and incubated for 1 hour at 37 °C. After washing, the plates were developed with 3,3′–5,5′-tetramethylbenzene (BM Blue POD substrate; Roche Applied Science, Indianapolis, IN); the reaction was stopped by addition of 1M H₂SO₄ and the optical density was read at 450 nm using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, Ca). The reported titers correspond to the reciprocal of the highest serum dilution showing a three times higher OD value than background.

SVs (10–20 µg) from each animal were loaded into 4–12% Bis-Tris wells (Invitrogen, Waltham, MA, USA) under reducing conditions, followed by transfer to a nitrocellulose membrane using iBlot2 (Invitrogen) and immunodetection. Nonfat milk (5%) was used to block nonspecific antibody binding (Thermo Fisher Scientific, Waltham, MA, USA). After blocking, membranes were incubated overnight at 4 °C with a primary antibody. Primary antibodies included GAPDH (Invitrogen), PSD95 (Invitrogen), GFAP (Sigma-Aldrich, St. Louis, MO, USA), SYP (ThermoFisher), VGLUT1 (Santa Cruz Biotech, Dallas, TX, USA), and SNAP25 (Synaptic Systems, Goettingen, Germany). MEGF8 (Bioworld Technology, St. Louis Park, MN, USA) and LAMTOR4 (Cell Signaling, Danvers, MA, USA) were additionally selected from the proteomic analysis for post-validation. Secondary antibodies were HRP-conjugated anti-rabbit IgG (Thermo Scientific) and HRP-conjugated anti-mouse IgG (Thermo Scientific). Primary and secondary antibody dilutions were done according to the manufacturer’s suggestion and are shown in Supplementary Table S1. Blots were developed using Azure cSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Whole cells were lysed in radioimmunoprecipitation (RIPA) lysis buffer [1% NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM phenyl-methylsulfonyl fluoride, and 0.25% sodium deoxycholate] containing protease inhibitor mixture (Sigma-Aldrich). The Bradford method was used for estimation of protein concentration of cell lysates. The equivalent amount of proteins (10 μg) in different groups were separated by 10% SDS-PAGE and transferred to PVDF membrane (Bio-Rad). The membranes were blocked for 1 h at room temperature in TBST buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.5) containing 5% nonfat dry milk, probed with indicated primary antibodies (Abs) at an appropriate dilution overnight at 4°C, washed three times with TBST and then incubated with secondary Abs for 1 h at room temperature. After additional three washes with TBST, the membranes were stained with Immobilon TM Western Chemiluminescent HRP Substrate (Millipore) and detected using an Image Quant LAS4000 system (GE Healthcare). Abs were diluted as follows: anti-Myc (Santa Cruz Biotechnology) at 1:2000, HRP-conjugated anti-mouse IgG (Thermo Scientific) at 1:5000. The results were the representative of three independent experiments.

Primary antibodies used in this study included: mouse anti-BrdU and mouse anti-β-catenin, BD Biosciences (San Jose, CA); rabbit anti-phospho-S6 ribosomal protein (Ser240/244) XP mAb, rabbit anti-phospho-mTOR (Ser2448) mAb, Cell Signaling (Danvers, MA); mouse anti- β-actin, Sigma-Aldrich (St. Louis, MO); rabbit anti-Sox9, Millipore (Bilerica, MA) and mouse anti-active β-catenin Milipore (Bilerica, MA). The secondary antibodies used were HRP-conjugated anti-rabbit IgG, Cell Signaling and HRP-conjugated anti-mouse IgG, Thermo Scientific (Cincinnati, OH). Dulbecco's Modifid Eagle Medium (DMEM), M199 and cosmic calf serum were from HyClone (Logan, UT). Tamoxifen, Alcian blue, human apo-transferin, hydrocortisone and sodium selenite were obtained from Sigma-Aldrich. Human epithelial growth factor was from Peprotech (Rocky Hill, NJ). Human insulin and 0.05% Trypsin-EDTA were from Invitrogen (Carlsbad, CA). Rapamycin (RAP) was purchased from LC Laboratories (Woburn, MA).