α phospho akt ser473

Primary antibodies used were: α-ubiquityl-PCNA (D5C7P; Cat# 13439), α-PCNA (PC-10; Cat# 2586), α-pan-Akt (Cat# 4691), α-phospho-Akt (Ser473; Cat# 9271), α-phospho-GSK3B (Ser9; Cat# 9336), α-phospho-PRAS40 (Thr246; Cat# 2997), α-RAD18 (Cat# 9040) and α-SMC-1 (Cat# 4802) from Cell Signaling Technology; α-BRCA1 (Ab-1) from Oncogene Research; α-PCNA (PC-10, Cat# sc-56) from SCBT; α-γH2AX (Cat# 05-636-1) from Millipore; α-CPD (Cat# NMDND001) from Cosmo Bio; α-Tubulin (Cat# T9026) from Sigma-Aldrich. Secondary antibodies used were: α-mouse Alexa Fluor 594 from Jackson ImmunoResearch; goat α-mouse IRDye 680RD (Cat# P/N 925-68070) and goat α-rabbit IRDye 800CW (Cat# P/N 925-32211) from LI-COR Biosciences. Nuclei were stained with DAPI (Cat# D9542) from Sigma-Aldrich.

Whole-cell lysates were prepared in lysis buffer (1% SDS, 10% glycerol, 80 mM Tris-HCl, pH 6.8) that had been pre-heated to 95°C. Proteins were transferred to Immobilon-FL PVDF membranes (Millipore) in transfer buffer (50 mM Tris base, 40 mM glycine, 0.04% SDS, 10% methanol) with an Owl semidry transfer apparatus (Thermo Scientific). Membranes were incubated with primary antibodies overnight at 4°C in 3% skim milk in Tris-Buffered Saline Tween-20 (TBST) (50 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20 [pH = 8.0]). Primary antibodies were α-phospho-AKT (Thr308) (#2965, Cell Signaling, 1:1000), α-phospho-AKT (Ser473) (#4060, Cell Signaling, 1:1000), α-AKT (#2920, Cell Signaling, 1:1000), α-phospho-S6 (Ser240/244) (#5364, Cell Signaling, 1:1000), α-S6 (#2317, Cell Signaling, 1:1000), α-SOX2 (MAB2018, R&D systems, 1:500), and α-SHP2 (C-18, Santa Cruz, 1:2000). After primary antibody binding, membranes were washed three times with TBST and probed with anti-rabbit (IRDye 800RS, LI-COR Biosciences, 1:10,000) and goat anti-mouse (Alexa Fluor 680, Invitrogen, 1:15,000) secondary antibodies in TBST for 1 hour at room temperature. After washing three times in TBST, proteins were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences).

Cells were treated with the 5o (1 µM for Huh7 and 4 µM for Mahlavu), 5m (1 µM for Huh7 and Mahlavu) and with DMSO as control for 72 h. After 72 h incubation, the cells were collected with scraper, their total proteins were isolated and protein concentrations were calculated with Bradford assay. Bio-Rad protein electrophoresis (Mini-PROTEAN^® TetraCellSystems and TGX™ precast gels, Bio-Rad, Hercules, CA, USA) and transfer system (Trans-Blot^® TurboTransfer System, Bio-Rad, Hercules, CA, USA) were used according to the manufacturer’s protocol for all the Western blotting analyses. About 20–40 µg of protein were used per well. Proteins were transferred to a PVDF membrane. For immunoblotting, PARP (#9532S, Cell Signaling), p21/WAF1/Cip1 (#05-345, Millipore), p53 (#05-224, Millipore), phospho-p53^Ser15 (#9286S, Cell Signaling), Rb (#9309, Cell Signaling), and phospho-Rb^Ser807/811 (#9308S, Cell Signaling), α-phospho-Akt^Ser473 (Cell Signaling, #9271), and AKT (#9272, Cell Signaling) antibodies were used in 1:100 to 1:500 5% BSA-TBS-T. β-actin (#A5441, Sigma) antibody was used in 1:1000 concentration for equal loading control. Proteins were visualized using a C-Digit^® imaging system (Ll-COR)