Protein a g bead

DF1 cells were seeded into 6-well plates and cultured in monolayers to 80%–90% confluence prior to co-transfection with 2 μg p∆CSGalNAcT2 and 2 μg pGtVP2 or 2 μg empty vectors as controls, using Lipofectamine 2000 (Invitrogen). At 36 h post-transfection, the transfected cells were washed three times with cold PBS and the cell lysates were prepared by adding 200 μL western and immunoprecipitation lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) containing 1 mM PMSF (Beyotime Institute of Biotechnology). The clarified lysates were precleared with protein A/G beads (Santa Cruz, Dallas, TX, USA); after centrifugation, 40 μL supernatant was used as the input, and another 160 μL supernatant was incubated with the anti-VP2 monoclonal antibody (mAb) [35 ] for 2 h at 4 °C, before adding protein A/G beads for overnight at 4 °C. Finally, the beads complexes were washed three times with cold PBS, boiled in 5 × sample loading buffer for 10 min, and subjected to electrophoresis on 12% SDS-PAGE gels, followed by immunoblot analysis with either the anti-FLAG (Sigma, St Louis, MO, USA) or the anti-VP2 mAbs [35 ], and then IRDye^® 800CW goat anti-mouse IgG (LiCor BioSciences, Lincoln, NA, USA). Blots were detected using an Odyssey Infrared Imaging System (LiCor BioSciences, Lincoln, NA, USA).

Cells were lysed in the lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 0.5% Igepal CA630, 5 mmol/L MgCl₂ and protease/phosphatase inhibitors (Yeasen). Lysates were passed through 30gauge needles 20times and centrifuged at 14,000 g for 10 min at 4ºC. The supernatants were collected and pre-cleaned with protein A/G beads (Santa Cruz Biotechnology) for 90 min at 4ºC. Lysates were centrifuged briefly, and the supernatants were collected while the protein A/G beads were discarded. Primary antibody was added to cell lysates and incubated at 4ºC overnight. protein A/G beads were then added to the cell lysates and incubated for 2 h at 4ºC. The beads were washed with cold lysis buffer 3 times. 1.5x SDS loading buffer containing 5% β-mercaptoethanol was added, and the samples were boiled for 5 min.

Cells were lysed with NP40 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 1× protease inhibitor) or modified NP40 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 1 mM MgCl₂, 250 U/ml benzonase (Sigma, E1014), 1× protease inhibitor; aims to degrade DNA) for 30 min on ice. After centrifugation at 13,400 × g for 15 min at 4 °C, the supernatant was collected for precipitation with the indicated antibodies overnight at 4 °C, followed by incubation with Protein A/G beads (Santa Cruz, sc-2003) for 2 h at 4 °C (for immobilized antibodies, precipitate for 1 h at 4 °C and without further incubation with Protein A/G beads). The beads were then washed with BC100 buffer (20 mM Tris-HCl pH 7.3, 100 mM NaCl, 10% glycerol, 2 mM EDTA, 0.1% Triton X-100) three times, and the protein complexes were then eluted with competitive peptide, 2× Laemmli buffer or 0.1% TFA.

Plasmids encoding human or murine MAG-ECD-Fc, Tie2-ECD-Fc, ANGPTL2-strep II and control vector were transiently co-transfected into 293T cells. The supernatant were collected 48 h after transfection and incubated with Protein A/G beads (Santa Cruz, sc2003) at 4 °C for 8 h, followed by washing with pre-chilled PBS with 0.1% NP-40 for 5 times. In the co-immunoprecipitation experiment for FYN and MAG, 293 T cells were transiently co-transfected with MAG (full-length)-FC, FYN-strepII and control vectors, lysed and incubated with Protein A/G beads (Santa Cruz, sc2003) or anti-strepII (GenScript, Cat#A00626-40) at 4 °C overnight, followed by washing with pre-chilled PBS with 0.1% NP-40 for 8 times. The immunoprecipitated proteins were examined by using the indicated antibodies for anti-strepII (GenScript, Cat#A00626-40) or anti-Fc (Sigma, Cat#12136) by western blot. The unedited agarose gel figures were showed in Additional file3