Hrp conjugated anti gapdh

SMAD2 and pSMAD2 protein expression was analyzed by western blot as previously described^{48 (link)}. Briefly, cell lysate samples containing 30 µg of total protein were separated and blotted using the Bio-Rad V3 Western Workflow system according to the manufacturer’s recommendation. Immunoblotting was performed using Phospho-SMAD2 (Ser465/Ser467), cat# E8F3R Rabbit mAb and anti-SMAD2, cat# D43B4 XP® Rabbit mAb, cell signaling technology (Danvers, MA, USA). The primary antibody was incubated overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit was used as the secondary antibody, whereas HRP-conjugated anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (1:1000; Invitrogen) was used as a loading control. Chemiluminescent detection was performed using WesternSure Chemiluminescent Substrate (LI-COR, Lincoln, NE, USA).

Cells were lysed using RIPA buffer (Pierce Inc, Thermo Scientific, USA) containing 1 × Halt Protease Inhibitor Cocktail (Pierce Inc., Thermo Scientific). Samples containing 30 µg of total protein were electrophoretically separated and blotted using the Bio-Rad V3 Western Workflow system according to the manufacturer’s recommendation. Immunoblotting was performed using anti-LIN28B rabbit monoclonal antibody (Ab191881, 1:2000; Abcam, Cambridge, MA). The primary antibody was incubated overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit was used as the secondary antibody, whereas HRP-conjugated anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (1:1000; Invitrogen) was used as a loading control. Chemiluminescent detection was performed using WesternSure Chemiluminescent Substrate (LI-COR, Lincoln, NE, USA). Band intensities were quantified using the band quantification tool in Image Laboratory 5.0 software (Bio-Rad Laboratories).