F7425

The procedure of protein extraction from Arabidopsis seedlings, separation, detection with antibodies, and quantification are described as previously^{46 (link)}, except 60 µg total protein were used for immunoblotting. The primary antibodies used for detection are: for GI-HA, anti-HA-Biotin antibody (1:1000, 12158167001, Millipore-Sigma); for ZTL, anti-ZTL antibody^{16 (link)} (1:200); for TMG, anti-GFP (1:10000, ab-290, Abcam); for FLAG-ZTL decoy, anti-FLAG antibody (1:1000, F7425, Millipore-Sigma). To quantify expression levels, the levels of target proteins were normalized to actin (anti-Actin antibody, 1:2000, SAB4301137, Millipore-Sigma).

The following primary antibodies were used in this study: mouse anti-FLAG (F3165, MilliporeSigma), rabbit anti-FLAG (F7425, MilliporeSigma), rabbit anti-pY654-β-cat (ab59430, abcam), rabbit anti-Src (701396, Invitrogen), mouse anti-phosphotyrosine (clone PY20, P4110, MilliporeSigma), mouse anti-SHP1 (MA5-11669, Invitrogen), rabbit anti-β-cat (clone RM276, SAB5600086, MilliporeSigma), mouse anti-β-cat (610153, BD Bioscience), mouse anti-γ-catenin (610253, BD Bioscience), rabbit anti-AXIN2 (PA5-25331, Invitrogen), mouse anti-GAPDH (CB1001, MilliporeSigma).

Embryos were manually deyolked and harvested at the time points indicated. Lysates were generated as previously described (21 (link)). Western analyses for Ctsk transgenes were performed using an anti-Flag antibody (MilliporeSigma, F7425, 1/800) and HRP-conjugated secondary antibody. Blots were analyzed using the Bio-Rad MP Chemidoc system. Total protein load as assayed by Ponceau S staining was used to ensure equivalence of loading between lanes.

Antibodies used in this study include rabbit anti-Syncytin-1 (for western blot, PA522819; Invitrogen), rabbit anti-Syncytin-1 (for flow cytometry, PA522819; Invitrogen), mouse anti-bZIP (sc69797; Santa Cruz), mouse anti-K8.1 (sc65446; Santa Cruz), rabbit anti-KSHV RTA (a gift from Yoshihiro Izumiya at University of California Davis), mouse anti-β-actin (AC-15; Sigma), rabbit anti-GAPDH (2118S; Cell Signaling), mouse anti-EA-D (MAB8186; EMD), mouse IgG1 negative isotype (CBL610; EMD Millipore), mouse anti-ZEBRA (a gift from Paul Farrell at Imperial College London, London, United Kingdom), rabbit anti-FLAG (F7425; Millipore), rat anti-EBNA2 (MABE8; Millipore), mouse anti-BrdU (555627; Thermo Fisher Scientific), mouse anti-CD19-APC (MABF197; Sigma-Aldrich), mouse anti-CD20-PE (MABF1635; Sigma-Aldrich), mouse IgG1-APC isotype (550824; BD Pharmingen), rat IgG2a isotype (MABF1077Z; EMD Millipore), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) (AP308P; EMD Millipore), HRP-conjugated goat anti-rabbit IgG (AP307P; EMD Millipore), FITC-conjugated goat anti-mouse IgG (F0257; Sigma), and APC-conjugated goat anti-rabbit IgG. All antibodies were used at concentrations and conditions recommended by manufacturers.

Exponentially growing yeast cells were harvested, washed with 2×HC buffer (300 mM HEPES-KOH pH 7.6, 2 mM EDTA, 100 mM KCl, 20% glycerol, 0.1% NP-40, 2 mM DTT and protease inhibitor cocktail (Roche)) and frozen in liquid nitrogen. Crude cell extracts were prepared by vigorously blending frozen yeast cells with dry ice in a household blender, followed by sonication and incubation with 30 ml 1×HC buffer containing 250 mM KCl for 30 min. The lysate was cleared by centrifugation at 82 700 × g for 3 h. The supernatants were incubated with 150 μl of Flag antibody agarose beads, washed eight times with 1×HC containing 250 mM KCl, then two times with the same buffer without NP-40. For mass spectrometry analysis, bound proteins were eluted with 2 × 400 μl of 50 mM Tris pH 7.5, 5% SDS, 5% glycerol and 50 mM DTT for Cwf14-Myc purifications and with 200 mg/ml 3×Flag peptides for Flag-tagged protein purifications. For co-immunoprecipitation analyses, bound proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blot analyses with Myc (Santa Cruz Biotechnology, sc-789) and Flag (Sigma, F7425) antibodies.