Subcellular protein fractionation kit for cells

Cells were grown to near confluence, collected, and fractionated into subcellular components (Subcellular Protein Fractionation Kit for Cells, ThermoScientific, Rockford, IL, 61101). The subcellular fractions were run on a precast polyacrylamide gel (BioRad, Hercules, CA, 94547), along with Full-Range Molecular Weight Rainbow Markers (GE Healthcare Life Sciences, Piscataway, NJ, 08855) then transferred to a PVDF membrane. Western analysis was performed on the membrane with a rabbit polyclonal antibody against human nucleolin (Abcam, Cambridge, MA, 02139), and ECL Plex goat anti-rabbit IgG Cy5 secondary antibody (GE Healthcare Lifesciences, Piscataway, NJ, 08855). The blot was analyzed on a Typhoon 9410 variable mode imager (GE Healthcare Lifesciences, Piscataway, NJ, 08855) and quantified.

Fractionation of subcellular proteins was done by using Subcellular Protein Fractionation Kit for cells (Thermo Fisher Scientific Inc.). Western blot analysis was performed by standard methods. Results shown are representative of at least two independent experiments. Antibodies specific for cleaved caspase-3 (#9664), cleaved PARP (#5625), and p21CIP1 (#2947) were from Cell Signaling; antibody for STAMP2 (11944-1-AP) and ATF4 (10835-1-AP) was from Proteintech Group Inc.; and antibodies for PCNA (P8825) and GAPDH (G8795) were from Sigma. Antibody for STAMP1 was described previously (Wang et al, 2010 (link)). All antibodies were used at a dilution of 1:1,000, except for GAPDH (1:5,000).