Criterion tgx stain free precast polyacrylamide gels

For whole-cell radioimmunoprecipitation assay (RIPA) lysates, cell pellets were lysed in 1 volume of RIPA buffer [150 mM NaCl, 5 mM EDTA, 50 mM tris (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] in the presence of Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, #1861282) for 15 min on ice and centrifuged at maximum speed for 15 min at 4°C. The protein concentration of the lysate was measured using the Pierce PCA Protein Assay Kit (Thermo Fisher Scientific, #23225), and equal amount of proteins (10 to 30 μg) was separated by 4 to 15% Criterion TGX Stain-Free precast polyacrylamide gels (Bio-Rad, #5678084). After transfer on nitrocellulose membranes, we detected our protein of interest using the following antibodies: INTS1 (Bethyl Laboratories, #A300-361A), INTS3 (Sigma Prestige, #HPA074391), INTS6 (Novus Biologicals, #NB10086990), INTS7 (Bethyl Laboratories, #A300-271A), INTS11 (Sigma Prestige, #HPA029025), glyceraldehyde-3-phosphate dehydrogenase (Abcam, #ab8245), DROSHA (Abcam, #ab12286), DICER (Abcam, #ab14601), DGCR8 (Abcam, #ab90579), AGO1 (Cell Signaling, #D84G10), AGO2 (Abcam, #ab57113), AGO3 (Sigma-Aldrich, #SAB4200112), AGO4 (Cell Signaling, #D10F10), and lamin B1 (Proteintech, #66095-1-1g). Antibodies were used in 1:1000 dilutions.