Protease inhibitors cocktail

Total lysates from tissues or cells were obtained by lysing in RIPA buffer with protease inhibitors cocktail (#HY-K0010, MedChem Express, Shanghai, China). Protein concentration was measured by the BCA assay (Bio-Rad, Hercules, CA, USA). Proteins were extracted and separated in 10% Tris glycine/SDS–polyacrylamide gels and transferred to PVDF membranes (#IPFL00010, Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk and incubated with specific antibodies overnight at 4 °C. β-actin (Proteintech, #66009-1-Ig) was used as the endogenous control. Primary antibodies were used at the dilution of 1:1000. Anti-SCD1 (#2794), cyclin D1 (#2978), cyclin D2 (#3741), cyclin D3 (#2936), cyclin E1 (#4129), CDK4 (#12790), CDK6 (#13331), CDK2 (#2546), P18 (#2896), P21 (#2947), P27 (#3686), P-Rb (#8516) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-RASAL1 (ab168610) was obtained from Abcam (Cambrdige, MA, USA). Blots were then incubated with relevant secondary antibodies, HRP-conjugated Goat Anti-Rabbit IgG (SA00001-2) and HRP-conjugated Goat Anti-mouse IgG (SA00001-1) were purchased from Proteintech (Wuhan, China) for 1 h. Bands were detected with the enhanced chemiluminescence detection systeme (P10200, New Cell & Molecular Biotech Co., Ltd) and Bio-Rad ChemiDocTM MP imaging system. Relative abundance was measured with Image J software.

Total lysates from HUVEC cells were obtained by lysing in RIPA buffer with protease inhibitors cocktail (#HY-K0010, MedChem Express, Shanghai, China). Protein concentration was measured by the BCA assay (Bio-Rad, Hercules, CA, USA). Proteins were extracted and separated in 10% Tris glycine/SDS–polyacrylamide gels and electro-transferred to ECL nitrocellulose membranes (#IPFL00010, Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk and incubated with specific antibodies overnight at 4 °C. β-Actin or P85 was used as the endogenous control. Primary antibodies were used at the dilution of 1:1000. Anti-Phospho-Akt (Ser473) (#4060), Akt (#4685), PI3K (#4249), stathmin1 (#13655) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin (ab8266), anti-P85 (ab191606), anti-α Tubulin (acetyl K40) (ab24610) and horseradish peroxidase-conjugated anti-mouse or rabbit IgG were purchased from Abcam (Cambridge, MA, USA).