Biospectrum 810 imaging system

GST pulldowns and immunoprecipitations with glutathione-sepharose beads or protein G-sepharose beads (GE Healthcare Life Sciences), respectively, were carried out as described [4] (link), [18] (link). Recovered proteins and cell extract (30 µg of protein) were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted as described [4] (link), [18] (link). Immunoblotted membranes were imaged with a UVP Biospectrum 810 Imaging System (Upland, CA) and analyzed with VisionWorksLS software. Differences in the levels of specified proteins between two cell samples were quantified by comparing protein band intensities normalized to actin, and the Student's t-test was performed to determine statistical significance. E4-ORF1-binding proteins were identified by conducting a pulldown assay with extracts of suspension-cultured HeLa cells, resolving recovered proteins by SDS-PAGE, digesting separate gel sections with trypsin, and subjecting the released peptides to MALDI-TOF mass spectrometry.

Western blot analysis was performed as previously described (Chakraborty & Ain, 2017 (link)). Total protein was extracted from trophoblast cells using RIPA buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 0.2 mM PMSF, and 1 mM sodium orthovanadate) containing Protease Inhibitor Cocktail (Sigma-Aldrich). A Bio-Rad protein assay reagent (Bio-Rad) was used to estimate the concentration of each protein sample. Then, 80–100 μg of protein extracts were fractionated using 10–12% SDS–PAGE under reducing condition and were then transferred onto to PVDF membranes (Millipore). The membranes were then blocked with 5% skim milk in TBS-T for 1 h at room temperature. Primary antibodies were diluted in milk or BSA as per the manufacturer’s instructions and incubated with the membranes at 4°C overnight. Secondary antibodies were diluted in TBS-T and incubated with the membranes at room temperature for 1 h 30 min. An ECL reagent, Luminata Forte (Millipore), was used for chemiluminescence signal detection. Images were acquired with the BioSpectrum 810 imaging system (UVP), and band intensities were quantified using NIH ImageJ software (https://imagej.nih.gov/ij/) and normalized to RPL7 for each sample. Each experiment was performed in triplicates using different biological replicates.

Western blotting was performed as described previously^{61 (link)}. Total protein was extracted from tissue or cells using RIPA buffer. Protein concentration for each sample was estimated by using the Bio-Rad Protein Assay reagent (Bio-Rad, USA). 80–100 µg of total proteins were separated by 10–12% SDS-PAGE under reducing condition and were then transferred to PVDF membranes (Millipore, USA). The blots were then blocked for 1 hour in 5% skim milk in TBS-T. Rabbit anti-TTR (Santa Cruz Biotechnology) and Rabbit anti-RPL-7 (Bethyl Lab) antibodies were used at recommended dilutions. The blots were incubated in primary antibody solution for overnight at 4 °C. Then the blots were incubated in HRP-conjugated goat anti-rabbit IgG (Cell signaling Technology) diluted 1:2000 in TBS-T for 1.5 hours at room temperature. Blots were developed using an ECL kit (Millipore) according manufacture’s protocol and images were captured by Biospectrum 810 imaging system (UVP, USA). Densitometric analysis was done by NIH ImageJ software. Three to five biological replicates were used for each experiment.