Complete ultra mini tablet

For Western blot analysis, cells were lysed in RIPA buffer (20 mM Tris pH 8.0, 0.1% SDS, 150 mM NaCl, 0.08% sodium deoxycholate, and 1% NP40 supplemented with 1 tablet of protease inhibitor (Complete ultra mini-tablet, Roche, Indianapolis, IN, USA) and phosphatase inhibitor (PhosStop tablet, Roche, Indianapolis, IN, USA)). A total of 20 µg of total protein was loaded per lane, and protein was separated using SDS-PAGE. The separated proteins on the gel were transferred onto a PVDF membrane and were probed for specific antibodies against Rb (cat#9390; Cell signaling, Danvers, MA, USA), phospho-Rb (cat#8516, Cell signaling, Danvers, MA, USA), E2F2 (ab209662; Abcam, Cambridge, UK), HK1(cat#2024; Cell signaling, Danvers, MA, USA), and GAPDH (cat#5174; Cell signaling, Danvers, MA, USA) at 1:1000 dilution in 5% BSA in 1× TBST, overnight at 4 °C. After 4 washes with 1× TBST for 10 min, membranes were incubated with HRP-conjugated anti-mouse (cat#7076; Cell signaling, Danvers, MA, USA) or anti-rabbit antibodies (cat#7074; Cell signaling, Danvers, MA, USA) at 1:2000 dilution for 2 h. Images were visualized using the Image Quant LAS 500 system (GE Healthcare Life Sciences, Piscataway, NJ, USA).

For Western blot analysis, cells were lysed in RIPA buffer (20 mM Tris pH 8.0, 0.1% SDS, 150 mM NaCl, 0.08% sodium deoxycholate, and 1% NP40) supplemented with 1 tablet of protease inhibitor (complete ultra mini-tablet, Roche, Indianapolis, IN, USA) and phosphatase inhibitor (PhosStop tablet, Roche, Indianapolis, IN, USA). A total of 20 µg of total protein was loaded per lane and separated by SDS-PAGE. The separated proteins on the gel were transferred onto the PVDF membrane and were probed for specific antibodies against Rb (cat#9309; Cell Signaling Technology, Danvers, MA, USA), phospho-Rb (cat#8516, Cell Signaling Technology, Danvers, MA, USA), E2F2 (ab209662; Abcam, Cambridge, UK), HK1(cat#2024; Cell Signaling Technology, Danvers, MA, USA) and GAPDH (cat#5174; Cell Signaling Technology, Danvers, MA, USA) at 1:1000 dilution in 5%BSA in 1× TBST overnight at 4 °C. After 4 washes with 1× TBST for 10 min, membranes were incubated with HRP-conjugated anti-mouse (cat#7076; Cell Signaling Technology, Danvers, MA, USA) or anti-rabbit antibodies (cat#7074; Cell Signaling Technology, Danvers, MA, USA) at 1:2000 dilution for 2 h. Images were visualized using the Image Quant LAS 500 system (GE Healthcare Life Sciences, Piscataway, NJ, USA).