Immobilon western hrp substrate luminal reagent

Total protein was extracted from cell lines by RIPA lysis buffer (Cat No. CW2333S, CWBIO) supplemented with protease and phosphatase inhibitor cocktail (Cat No. CW2200S and CW2383S, CWBIO). Protein was quantified using the BCA Protein Assay Kit (Cat No. CW0014S, CWBIO) and denatured at 70°C for 10 mins with 5× SDS-PAGE loading buffer (Cat No. CW0027S, CWBIO). Equivalent amounts of proteins were separated by 6% or 8% SDS-PAGE. After electrophoresis, the proteins were transferred onto polyvinylidene fluoride membrane. Afterwards, membranes were blocked with 5% skim milk for 1 hr at room temperature and then incubated with the corresponding primary antibody at 4°C overnight. The next day, after being washed 3 times with PBST (phosphate-buffered saline with 0.1% Tween-20) buffer, and the membranes were incubated with the corresponding secondary horseradish peroxidase conjugated anti-rabbit or anti-mouse antibody at room temperature for 1 hr. Finally, Immobilon Western HRP Substrate Luminal Reagent (Cat No. P90714 and P90715, MILLIPORE) was employed for detection of protein bands using an enhanced chemiluminescence detection system (Amersham Imager 600, GE). Information on corresponding primary and secondary antibodies is listed in Table S2.

Total protein extracted from cells were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. After incubated with a series of primary antibodies at 4°C overnight, the membranes were incubated with secondary antibody for 1 h at room temperature. Immobilon Western HRP Substrate Luminal Reagent (WBKLS0500, MILLIPORE, Billerica, MA, USA) and an enhanced chemiluminescence detection system (Amersham Imager 600, GE) were used to detect protein bands. All primary and secondary antibodies are listed in Table 3.Table 3

The Information of Antibodies

Gene Name	Catalog Number	Company	Diluted Concentration	Secondary Antibody
β-actin	A4552	Sigma	1:20,000	Mouse
RRM2	ab57653	Abcam	1:500	Mouse
Akt	4691	Cell Signaling Technology	1:1000	Rabbit
mTOR	2983T	Cell Signaling Technology	1:1000	Rabbit
4EBP1	9644	Cell Signaling Technology	1:1000	Rabbit
PRAS40	2691	Cell Signaling Technology	1:1000	Rabbit
Phospho-Akt	4060	Cell Signaling Technology	1:2000	Rabbit
Phospho-mTOR	5536	Cell Signaling Technology	1:1000	Rabbit
Phospho-PRAS40	2997	Cell Signaling Technology	1:1000	Rabbit
Phospho-4EBP1	2855	Cell Signaling Technology	1:1000	Rabbit
Anti-mouse IgG	7076	Cell Signaling Technology	1:5000
Anti-rabbit IgG	7074	Cell Signaling Technology	1:5000