Anti erk1 2

TRIzol reagent and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The First Strand cDNA Synthesis kit and SYBR Premix Taq were from Takara (Dalian, Liaoning, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), bromodeoxyuridine (BrdU) and the anti-BrdU antibody were purchased from Sigma (St. Louis, MO, USA). DAPI, BCA protein assay and ECL Plus kits were obtained from Beyotime Institute of Biotechnology (Beijing, China). BD BioCoat Matrigel invasion chambers were purchased from BD Biosciences (San Jose, CA, USA).
The primary antibodies against human SKA1 and cleaved caspase-3 were obtained from Abcam (Cambridge, MA, USA). Anti-Bcl-2, anti-Bax, anti-p-ERK1/2, anti-ERK1/2, anti-p-Akt, anti-Akt, anti-p21, anti-cyclin D1 and anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology. Biotinylated- and Cy3-conjugated anti-rabbit secondary antibodies were purchased form Boster (Wuhan, Hubei, China).

Cells were lysed for 30 min in Triton buffer (1% Triton X-100, 50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (1 mM PMSF, 2 mM sodium pyrophosphate, 2 mM sodium betaglycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Lysates were cleared by centrifugation at 15,000 × g at 4°C for 15 min, and protein concentrations were determined via the Bradford method. 50 μg of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes. Proteins were detected by using anti-LETM2 (Proteintech, 17180-1-AP), anti-FGFR1 (Abcam cat# ab76464; RRID: AB_1523613), anti-WHSC1L1 (Abcam, ab180500), anti-CDCA7 (Abcam cat# ab69609; RRID: AB_1268064), anti-ERK1/2 (Santa Cruz, sc-514302), anti-p-ERK1/2 (Cell Signaling Technology cat# 4376; RRID: AB_331772), anti-AKT1 (Cell Signaling Technology cat# 2967; RRID: AB_331160), and anti-p-AKT1 (Cell Signaling Technology cat# 9018). Antibody binding was detected using horseradish peroxidase-labeled anti-mouse (Sigma) or anti-rabbit (Cell Signaling) antibodies and chemiluminescence was detected with a LAS4000 device (Fuji). Equal protein loading was confirmed with antibodies against GAPDH (Transgen).