Anti cyclin d1 sc 8396

Human GC cell lines (AGS, BGC-803, BGC-823, MKN-45 and SGC-7901)
were purchased from the Type Culture Collection of the Chinese Academy of
Sciences (Shanghai, China). The human normal gastric mucosal cell line GES-1 was
acquired from Biowit Technologies Corporation (Shenzhen, China). All cell lines
were cultured as described previously [14 (link)].
Mouse anti-cyclin B1(sc-245) and anti-Cyclin D1 (sc-8396) were
purchased from Santa Cruz (Santa Cruz, CA, USA), whereas mouse
anti-CDK4(#2906S)and anti-CDK6(#3136S)were purchased from Cell Signaling
Technology (CST, MA, USA). Rabbit anti-USP22 (55110–1-AP), anti-OCT1 (POU2F1)
(10387–1-AP), anti-E-cadherin (20874–1-AP), anti-γ-catenin (27872–1-AP),
anti-Vimentin (Ag0489), and anti-MMP2 (10373–2-AP) were purchased from
Proteintech (Wuhan, China). Rabbit anti-ERK1/2 (137F5), anti-p-ERK1/2
(Thr202/Tyr204), anti-AKT (C-terminal), and anti-p-AKT (Ser473) were purchased
from CST (MA, USA). Rabbit anti-GAPDH and anti-Ki-67 (EPR3610) were purchased
from Abcam (Abcam, Cambridge, UK).

Total cell lysate was prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS and a mixture of protease inhibitors). Western blot analysis was performed using the same amount of protein. Protein concentration was determined by the Bradford (Sigma-Aldrich, St. Louis, MO, USA) method and equal quantities were subjected to Western blot analysis. SDS-PAGE-separated proteins were electroblotted onto a nitrocellulose membrane. The blots were incubated overnight at 4 °C with the following primary antibodies: anti-ERRα (ab76228; 1:1000); anti-SR-BI (ab52629; 1:1000); anti-HMGCR (ab215365; 1:500); from Abcam, Cambridge, UK; anti-Cyclin D1 (sc-8396; 1:500), anti-PARP-1 (sc-7150; 1:2000) and anti-GAPDH (sc-32233; 1:10,000) from Santa Cruz Biotechnology, Dallas, TX, USA. Anti βactin (ab8226; 1:1000; Abcam) was used as a loading control. All antibodies were incubated with appropriate horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were detected by the ECL Western blotting detection system (Santa Cruz Biotechnology, sc-2048). All the whole western blot figures can be found in the Supplementary Materials.