Western Blotting. Samples were prepared using RIPA buffer (1 % Nonidet P-40, 0.5 % deoxycholate, 0.1 % SDS in PBS) containing a protease inhibitor cocktail (Roche), and loading buffer (20 % glycerol, 1M Tris HCl pH 6.8, bromophenol blue) to contain an equal amount of protein (20 µg). 15 µl of each sample was loaded and subjected to electrophoresis on 10 % SDS polyacrylamide gels (Biorad, Hercules, CA, USA). Protein standard markers (Bio-Rad Laboratories) were loaded and run parallel to each blot as an indicator of the molecular weight. The gels were run in running buffer (25 mM Tris, 192 mM glycine, 1 % w/v SDS) at 100 Volts for 1 hour and 20 minutes. The proteins were then transferred onto a 0.45 µm nitrocellulose membrane (Sigma-Xxxxxxx, USA) using transfer buffer (25 mM Tris, 192 mM glycine) at 30 Volts for 1 hour. After transfer, non-specific protein binding was blocked by incubation of the membrane with 5% non-fat dry milk in TBS-T buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1 % Tween-20) for 1 hour at room temperature. This was followed by incubation with primary antibodies: rabbit FGFR1 (1:500; Abgent, Beverly, MA, USA) and β-actin (1:2500; Sigma) in milk/TBS-T overnight at 4 °C with rocking. The immunoblots were then washed 3 times with TBS-T and incubated with a secondary antibody, goat anti-rabbit HRP (1:1000; DakoCytomation) to detect binding of the primary antibody for 1 hour at room temperature with gentle shaking. The immunoblots were developed and visualised using the Enhanced chemiluminescence (ECL) system (Amersham Hyperfilm™ ECL) and a Kodak Image station (Kodak Digital Science). When blots for β-actin were not performed in parallel to the target, the target blots were stripped using Re-Blot Plus Strong Solution (10X; Millipore) and re-probed. This protocol was also followed for C3 (1:100; Hycult biotech) and C5 (1:100; Hycult biotech) detection. The secondary antibody for both primary antibodies was rabbit anti-mouse HRP (1:1000; DakoCytomation).
Western Blotting. E12.5 embryos were dissected in PBS, and weighed to determine the amount of lysis buffer (250mM sucrose, 20mM pH7.9 Tris, 0.45M NaCl, 2mM MgCl2, 2mM CaCl2, 1% Sodium cholate, and protease inhibitor) that needed to be added. 1.5ml of lysis buffer was added to 1g of embryo weight, and tissue was homogenized by using a pestle on ice. Homogenized tissue was incubated on ice for 20 minutes, and protein was obtained by centrifuge at 58000 rpm for 1 hour at 4C. The supernatant was transferred to a new tube, and protein concentration was determined by Bradford assay. 50 g of protein was loaded to a 10% SDS-PAGE gel, and the proteins were separated at 200 volts for 40 minutes. After transferring proteins to a nitrocellulose membrane at 100mA at 4C overnight, the membrane was blocked in 5% milk in TBST (0.1% Tween-20 in 1X TBS) for 1 hour. Affinity purified Arl13b antibody (1:1000) was incubated with the membrane at RT for 1 hour, and the membrane was washed with TBST for 5 minutes three times. Anti-rabbit HRP (1:5000, GE healthcare NA934) was incubated with the membrane for 1 hour, and proteins were visualized by ECL method (GE Healthcare, RPN2432). Actin antibody (1:5000, Sigma A5060) was used for a loading control.
Western Blotting. Unless otherwise stated, all solutions were prepared as described in table 2.2.
Western Blotting. Protein samples were reduced and denatured in Laemmli buffer, loaded into 4-20% Tris-Glycine gels (Bio-Rad) for SDS-PAGE, and then transferred to nitrocellulose membranes (Bio-Rad). Blots were blocked with 5% milk (in 50mM NaCl, 10mM HEPES, pH 7.3 with 0.1% Tween-20 (Sigma)) and incubated with primary antibodies overnight at 4°C. Flag-tagged GPR37L1 and K349N were detected with mouse HRP-conjugated anti-Flag (Sigma). Protein quantification was done using densitometry, performed with ImageJ software.
Western Blotting. Cells were lysed in RIPA buffer (consists of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EDTA , 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4 , 1 µg/ml leupeptin; Cell Signaling) supplemented with protease and phosphatase inhibitors (Sigma-Xxxxxxx). Total protein extracts (30 µg) diluted with 5X sample buffer and boiled for 5 minutes. Proteins were resolved using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with either 10% or 12% gels followed by blotting onto a nitrocellulose membrane. The nitrocellulose blot was incubated for 1 hr in 5% bovine serum albumin followed by an overnight incubation at 4°C with the primary antibody, diluted in 5% bovine serum albumin. Blots were probed overnight using the following antibodies from Cell Signaling: phospho-Thr389 p70S6K clone 1A5 at 1:750; total p70S6K (1:1000), p- S473 Akt-XP used at 1:1000, and polyclonal antibodies against total Akt (1:1000), p- Thr202/Tyr204 p42/p44 ERK1/2 (1:1000), total p42/p44 ERK1/2 (1:1000), and FOXM1 (D12D55) XP (1:1000). β-actin monoclonal AC-15 (Sigma-Xxxxxxx) at 1:10,000 was used as a loading control. Protein bands were detected using the Odyssey Imaging System (Li-Cor Biosciences; Lincoln, NE). Experiments were repeated three times to ensure reproducibility.
Western Blotting. Immunoblotting was performed using standard methods. Briefly, patient and control lymphoblastoid cells were lysed with a standard Triton X-100-based lysis buffer. The lysate protein concentrations were measured with the Bradford assay. Proteins were denatured by heating at 95°C for 3 minutes and separated by polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. To assess protein loading and transfer, the membrane was reversibly stained with Ponceau S. The membrane was blocked for one hour in blocking buffer (10 g dry milk, 200 µl Tween-20, and 100 ml PBS), probed with primary antibody (anti-FMRP 1a or anti-eIF4e) overnight, and probed for one hour with horseradish-peroxidase conjugated anti-mouse secondary antibodies. Proteins were detected by chemiluminescence (ECL, GE Healthcare, Piscataway, NJ).
Western Blotting. SDS-PAGE was performed as described in 2.9.2. Proteins were then transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare) via wet transfer for 1 hour at 80 V and 4°C. The Criterion Blotter apparatus was used in this study (Bio-Rad). The membrane was then blocked at room temperature for 30 minutes with 5% milk +TBS. Incubations with primary antibodies were carried out over night with blocking buffer at 4°C. Following this, the membrane was washed at room temperature three times (10 minutes each) in TBST. The secondary antibodies were then applied and incubated with the membrane at room temperature for 1 hour in TBST. The membrane was washed again at room temperature three times (10 minutes each) in TBST. The TBST was removed and the membrane was applied with ECL chemiluminescence reagent (Xxxxxx) and exposed to Amersham hyperfilm ECL (GE Healthcare) and developed with an X-ray film processor (JP-33 model, Jungwon Precision Industry).
Western Blotting. The gel was washed in transfer buffer following which it was stacked between layers of scotch pads, 3mm Whatmann filter paper and a PVDF membrane which had been activated by washing with 100% methanol before use. The stack was placed inside the transfer tank and filled with 1x transfer buffer with 20% methanol (Recipe given in Table 2.2). The tank was kept cool using distilled water and ice packs. A current of 400mA and 35V was applied for 1.5hours and once the transfer was complete the membrane was blocked in 5% dried milk in PBS with 0.1% tween (Thermo-Scientific, UK), with constant shaking for 1 hour at room temperature. The membrane was then transferred to primary antibody in 5% milk in PBS + 0.1% tween overnight at 4°C. The membrane was washed 3 times in PBS+0.5% tween and then incubated for 1 hour at room temperature in Horseradish-peroxidase (HRP) conjugated IgG antibody. The membrane was again washed three times in PBS+0.5% tween-20. Detection of HRP conjugated antibodies was carried out using SuperSignal West Femto ECL Western Blotting system (ThermoScientific,, USA). The membrane was incubated in the enhanced chemiluminescent (ECL) reagents for 2-3 minutes at room temperature, wrapped in a plastic sheet and immediately imaged using Bio-Rad Chemi-Doc XRS Molecular imager.
