Dynabeadstm protein g

For immunoprecipitation experiments, WTT-CHO cells were transiently transfected with pcDNA3.1 (+) empty vector (control) or vectors encoding HA-GPCRs in similar conditions to those used for AFM-SMFS experiments. Forty-eight hours post-transfection, cells were lysed in a lysis buffer (140 mM NaCl/2 mM EDTA/25 mM Tris, pH 7.4/0.5% DDM) supplemented with complete protease and phosphatase inhibitors (Roche). Protein extracts (~2 mg) were immunoprecipitated overnight at 4 °C with Dynabeads^TM Protein G (Invitrogen) precoated with an anti-HA antibody (BioLegend, 16B12 Clone) and proceeded to western-blot analysis as previously described^{33 (link)}. Proteins were detected with the anti-HA primary antibodies (Biolegend, 16B12 Clone) followed by antimouse Horse Radish Peroxidase (HRP)-conjugated secondary antibodies (TrueBlot, eB144Clone, Rockland) using enhanced chemiluminescence detection reagent (RPN2232 Prime, GE Healthcare).

Cells were lysed in Lysis Buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 5% glycerol) added Proteinase Inhibitor Cocktail (Cat.78444, Thermo Scientific, USA) freshly before use. The cell lysates were incubated with anti-CHOP (#2895S, Cell Signaling Technology), anti-TXNIP (#14715S, Cell Signaling Technology), or anti-NEDD4L (13690-1-AP, Proteintech) antibodies overnight. Protein A (Dynabeads^TM Protein A, Cat.10002D, Invitrogen) or Protein G (Dynabeads^TM Protein G, Cat.10004D, Invitrogen) were pre-cleaned with Lysis Buffer three times and used to precipitate the immune complex. The precipitates were washed with lysis buffer three times, separated by SDS-PAGE, and immunoblotted with indicated antibodies.

Cells were transfected with a pEF1α-HER3 plasmid for 24 h (hs), and cells were treated with MG132 (25 μM) for 5 h. Subsequently, cells were mixed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40) supplemented with protease inhibitors (Roche, Switzerland). Equal amounts of total proteins (500 μg) were incubated with the anti-HER3 antibody at a dilution of 1:250 at 4 °C for 8 h. Then, proteins were incubated with 10 μl Dynabeads ^TM Protein G (Invitrogen, USA) at 4 °C for 2 h to pull down the antibody-bound proteins. The mixtures were washed and boiled in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 12.5% β-mercaptoethanol). The pulled-down proteins were analyzed for ubiquitinylation by Western blotting.

Extracted proteins from MKN‐28 cells were mixed together with the primary antibody, which was slowly shaken at 4°C overnight. The DynabeadsTM Protein G (Invitrogen) were washed twice in PBS before being used to prepare a 50% protein G beads working solution that was then added into the abovementioned antigen–antibody mixture and slowly shaken at 4°C for 4 h. By magnetic separation, the clear liquid was discarded, and the protein G beads were harvested and washed, and the samples were separated by boiling for next experiment.

PASMC lysates were prepared in lysis buffer (20 mM Tris–HCl [pH 7.5], 100 mM KCl, 5 mM MgCl₂, and 0.5% NP-40) containing a protease inhibitor cocktail (#11697498001, Roche, Basel, Switzerland). The lysates were precleared with Dynabeads^TM Protein G (#10007D, Invitrogen, Carlsbad, CA, USA) at 4 °C with gentle rotation for 1 h. The precleared lysates were incubated with Dynabeads^TM Protein G coated with 2 μg each of rabbit anti-nucleolin antibody (#ab22758, Abcam, Cambridge, UK) or rabbit IgG (#2729, Cell Signaling Technology, Danvers, MA, USA) at 4°C for overnight. For DGCR8 IP and Ago2 IP, 5 μg of mouse anti-DGCR8 antibody (#60084-1-Ig, Proteintech, Rosemont, IL, USA) and mouse anti-Ago2 antibody (#ab57113, Abcam) were used, respectively. A reaction containing mouse IgG (#sc-2025, Santa Cruz Biotechnology, Dallas, TX, USA) served as a negative control. Unbound materials were washed off using NT2 buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM MgCl₂, and 0.05% NP-40). All collected protein complexes were eluted with 2X Laemmli sample buffer supplemented with β-mercaptoethanol and boiled. The boiled supernatants and input (2%) samples were resolved by SDS-PAGE and analyzed by immunoblotting with the anti-NCL antibody (#ab22758), anti-DGCR8 antibody (#60084-1-Ig), or anti-Ago2 antibody (#ab57113).

In the coimmunoprecipitation assays for EcR-A, EcR-B1, Usp and FruBM, constructs encoding each protein were overexpressed in S2 cells. Then, 5 µg of one of the pMT-HA-EcR-A-V5-His, pMT-HA-EcR-B1-V5-His or pMT-MYC-Usp-V5-His plasmid vectors and 5 µg of the pMT-FLAG-fruBM plasmid vector were transfected into S2 cells (5×10⁷ cells) using FugeneHD (Roche Diagnostics, Indianapolis, IN, USA), and protein expression was induced by addition of copper sulfate. Lysates were prepared by homogenizing in a cold lysis buffer [50 mM HEPES, pH 7.5, 100 mM NaCl, 50 mM ZnSO₄, 10 mM NaF, 0.2% NP40 and complete Protease Inhibitor (Roche)] for 1 h at 4°C, then incubated with rabbit IgG (I0500C, Invitrogen) or rabbit anti-Flag antibody (F7425, Sigma-Aldrich) in the aforementioned lysis buffer for 3 h at 4°C. The immuno-complexes were precipitated using Dynabeads^TM Protein G (10004D, Invitrogen) according to the manufacturer's instructions. Finally, the immuno-complexes were analyzed by western blotting with a primary antibody, anti-V5 (1:5000; 46-0705, Invitrogen), FruMale (Usui-Aoki et al., 2000 (link)), or mouse anti-Hsp70 (1:5000; H5147, Sigma-Aldrich), and, as a secondary antibody, with horseradish peroxidase (HRP)-conjugated, anti-rabbit or mouse IgG antibody (1:3000; Sigma-Aldrich).

DynabeadsTM Protein G (Invitrogen) were washed in PBS +/+ using a magnetic rack (BioRad) and incubated with 40 μg of antibody/sample overnight at 4°C while rotating. Beads were washed in PBS +/+, and beads were then incubated with cell lysates (150 μg) for 3 days at 4°C while rotating. Flow through was removed for analysis and beads washed in PBS +/+ and boiled at 65°C in 3× Laemmli sample Buffer and assessed by SDS-PAGE.