Bioimager 600 system

Cells were lysed in 1X Passive Lysis Buffer (PLB) (Promega) and quantified using Bradford reagent (Thermo Scientific). Cell number equivalent amount of the sample is then diluted in 5X Sample Loading Buffer (312.5 mM Tris-HCl, pH 6.8, 10% SDS, 50% glycerol, 250 mM DTT, 0.5% bromophenol blue) and heated for 10 mins at 95°C. Usually 1/5^th of the total amount from a 4cm² well was loaded for both the control and experimental samples. Following SDS-polyacrylamide gel electrophoresis of the extracts, proteins were transferred to PVDF nylon membranes. Membranes were blocked in TBS (Tris-buffered saline) containing 0.1% Tween-20 and 3% BSA. Primary antibodies were added in 3% BSA for a minimum 16 hr at 4°C. Following overnight incubation with antibody, the membranes were washed at room temperature thrice for 5 min with TBS containing 0.1% Tween-20. Washed membranes were incubated at room temperature for 1 hr with secondary antibodies conjugated with horseradish peroxidase (1:8000 dilutions). Excess antibodies were washed three times with TBS-Tween-20 at room temperature. Antigen-antibody complexes were detected with West Pico Chemi luminescent substrate using standard manufactures protocol (Perkin Elmer). Imaging of all western blots was performed using an UVP BioImager 600 system equipped with VisionWorks Life Science software (UVP) V6.80.

Renilla and firefly luciferase activities were measured using a Dual-Luciferase Assay Kit (Promega) following the supplier’s protocol on a VICTOR X3 Plate Reader with injectors (PerkinElmer). Before measurement of luciferase activity, cells were lysed with the 1X Passive Lysis Buffer (Promega). FL-normalized RL expression levels for reporter and control were used to calculate fold repression as the ratio of control to reporter-normalized RL values.
Western blotting analysis of indicated proteins was performed as described previously (Pillai et al., 2005 (link); Ghosh et al., 2015 (link)). Supplemental Table S3 lists antibodies used for Western blot and IP. Imaging of all Western blots was performed using an UVP BioImager 600 system equipped with VisionWorks Life Science software, version 6.80 (UVP). Densitometric analysis of Western blot protein band intensities was quantified using ImageJ software.

RL and FL activities were measured using a Dual-Luciferase Assay Kit (Promega, Madison, WI) following the supplier's protocol on a VICTOR X3 Plate Reader with injectors (PerkinElmer, Waltham, MA). FL-normalized RL expression levels for reporter and control were used to calculate fold repression as the ratio of normalized control to reporter RL values. To avoid discrepancy in transfection efficiencies and avoid differential dilution of DNA cue to variable cell number, all transfections were performed in a single plate before splitting equal numbers of cells into plates of variable diameter to achieve LDC and HDC conditions.
Northern blotting of total cellular RNA (5–15 μg) was performed as described by Pillai et al., (2005) (link). For detection, ³²P-labeled 22-nt antisense DNA probes specific for respective miRNAs or siRL or U6 snRNA were used. Phosphorimaging of the blots was performed in the Cyclone Plus Storage Phosphor System (Perkin­Elmer), and Optiquant software (Perkin­Elmer) was used for quantification.
Western analyses of different miRNP components (AGO2, RCK/p54, and XRN1) were performed as described previously. Detailed list of antibodies used are given in Supplemental Table S3. Imaging of all Western blots was performed using an UVP BioImager 600 system equipped with VisionWorks Life Science software, version 6.80 (UVP, Cambridge, UK).