Reagents, antibodies and plasmids
Complete protease inhibitor was obtained from Roche (Basel, Switzerland). All restriction enzymes and Taq polymerase were obtained from TakaRa (TakaRa, Japan). Monoclonal ANTI-FLAG® M2 antibody, monoclonal ANTI-FLAG® M2-Cy3 antibody, and monoclonal Anti-c-Myc-Cy3 antibody were obtained from Sigma-Aldrich (St Louis, MO). c-Myc monoclonal antibody was obtained from Clontech (Palo Alto, CA). Mouse IgG was obtained from ZhongShan Goldenbridge Biotechnology (China). Cy5-conjugated goat anti-rabbit IgG was labeled in the lab. CSS-100 silylated slides (Aldehyde) were obtained from CEL Associates, Inc. (CEL Associates Inc., Pearland, Texas, USA). Genes for proteins with suspected interaction partners were cloned into pFLAG-CMV-2 and pCMV-myc expression vectors for transient expression in mammalian cells. ORF of different proteins contained the same protein domains as used for the YTH.
Cell culture and transfection
Human embryonic kidney HEK293 cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Hyclone), penicillin, streptomycin and glutamine. One day before transfection, 0.75~3 × 104 cells in 100 μl DMEM without antibiotics were plated in the 96-microwell format, so that cells will be ~90% confluent at the time of transfection. Total of 200 ng DNA constructs with half of each plasmid were transfected. Transfections were performed with Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer's instructions.
Preparation of aldehyde slides spotted with antibodies
CSS aldehyde slides (CEL Associates Inc., Pearland, Texas, USA) were spatially separated onto 3 × 6 frames by a removable waterproof stick-film containing 3 × 6 wells. The waterproof stick-film is manually sticked on the surface of the slide before used. Antibody printing onto the slides was performed by a Smart Arrayer-48 spotting robot (CapitalBio, Beijing, China), mounted with an ArrayIt micro spray pin from TeleChem (Sunnyvale, CA, USA). 1.0 mg/ml anti-flag antibody and 1.0 mg/ml normal murine IgG (both diluted with 1.0 mg/ml BSA in TBST) were printed in spots of 0.4 mm in diameter and 1.5 mm intervals (center to center) with an approximately 10 nL spotting volume. During spotting, humidity and temperature in chamber were maintained at 40% and 20°C, respectively. After the printing process, all slides were incubated overnight at 4°C to allow maximum binding of antibody to the aldehyde slide surface.
Traditional resin-based coimmunoprecipitation
For general cell lysis and co-immunoprecipitation of Flag-X and the candidate interactor Myc-Y, HEK293 cells were transfected with indicated expression vectors by Lipofectamine 2000. After 30 h, cells were harvested and lysed in EBC buffer [50 mM Tris-Cl (pH8.0), 120 mM NaCl, 0.5%(V/V) NP40, 1 mM EDTA] supplemented with 50 μg/ml PMSF and protease inhibitor cocktail (Roche, Basel, Switzerland) at room temperature for 10 min. After centrifugation (4°C, 12,000 rpm, 10 min) the supernatant was used immediately. Immunoprecipitations were performed using normal IgG (for preclear), anti-Myc and protein A/G-agarose (Santa Cruz, CA) at 4°C. The lysates and immunoprecipitates were detected by Western blot using the indicated primary antibodies and appropriate secondary antibody, followed by measurement with SuperSignal chemiluminescence kit (Pierce).
Miniaturized sandwich immunoassay of PPIs
HEK293 cells plated in 96-well dishes were manually transfected with indicated expression vectors with Lipofectamine 2000. After 30 h, cells were harvested and lysed in 20 μl EBC buffer, as described. The concentration of the cell lysates were determined by Bradford method. To make sure that the recombinant constructs are expressed in HEK293 cells, crude cell lysates were spotted onto the aldehyde slides by noncontact printing, and the expression level of bait and prey were detected on the slides using anti-FLAG-Cy3 and anti-myc-Cy3 respectively. The antibody spotted aldehyde glass slides were blocked by incubation with 10 mg/ml BSA in TBST [20 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.05% (V/V) Tween 20] at room temperature for 1 h. After blocking, slides were rinsed with TBST for 3 times. 20 μl of cell lysate was transferred into one frame on the slide surface and incubated for 2 h at room temperature. The slides were washed with TBST for 3 times to remove unbound proteins. Protein interacting partners were then detected with monoclonal anti-myc-cy3 (1:200) by incubation in a humid, dark chamber for 45 min. After 3 additional washes with TBST, the frames were removed and the slides were air-dried prior to imaging.
Signal detection and analysis
To collect the fluorescence signal in the MSIP, slides were scanned using a confocal laser fluorescence scanner Luxscan-10K/A (CapitalBio, Beijing, China) with a resolution of 10 μm per pixel. Laser power and photomultiplier gain were both set to 70%. Image analysis was carried out with Spot Data Pro 2.0 (CapitalBio, Beijing, China). The median pixel values of feature and background were used as the signal and background intensities, respectively. The net fluorescence intensity of each PPI was calculated as the fluorescence intensity of each bait (FLAG-fusions) and prey (myc-fusions) subtracted by the corresponding negative control (myc-tagged prey in the absence of FLAG-tagged bait).