Sc 8432

Primary antibodies against RORα (ab60134), E-cadherin (ab40772), vimentin (ab92547), Snail (ab53519), matrix metalloproteinase-9 (MMP-9) (ab38898), tissue inhibitor of metalloproteinase-3 (TIMP-3) (ab39184), Ki-67 (ab66155), and CD34 (ab81289) were provided by Abcam (Cambridge, MA, UK). Primary antibodies targeting β-catenin (sc-1496), p-β-catenin (sc-101650), Axin-1 (sc-14029), c-Jun (sc-44), c-Myc (sc-40), TCF-4 (sc-271287), and β-actin (sc-8432) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated secondary antibodies were provided by Abzoom (Dallas, TX, USA). Goat anti-rabbit IgG-HRP (KGAA35) and goat anti-mouse IgG-HRP (KGAA37) were provided by KeyGEN BioTECH Corp (Jiangsu, China). Goat anti-mouse IgG (H + L) was purchased from Protech Technology, Inc. (Rocky Hill, NJ, USA). pGL3-c-Myc promoter luciferase reporter plasmid was obtained from Guangzhou Cyagen Biosciences Inc.

Tissues or cells were lysed in RIPA buffer (Pierce, USA) supplemented with phosphatase inhibitors and protease inhibitors (Pierce, USA), and the protein concentration was quantified by the BCA kit (Pierce, USA). Soluble lysates (30 μg protein) were fractionated by 10% SDS-PAGE gels and transferred to nitrocellulose (NC) membranes (PALL, USA). The membranes were immersed in Tris-buffered saline-Tween 20 (TBST) containing 5% bovine serum albumin (BSA) to block non-specific background staining and then incubated at 4°C overnight with primary antibody. STIM1 (sc-68897), TGIF (sc-9084), Akt (sc-8312), cyclin A (sc-751), cyclin B1 (sc-752), CDK4 (sc-260), cyclin D1 (sc-718), p21 (sc-397), and β-Actin (sc-8432) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PTEN (#9188S), phospho-Rb (#8516S), p65 (#8242S), β-catenin (#9582S), and c-Myc (#13987S) were obtained from Cell Signaling Technology (Cell Signaling, USA). The membranes were washed with TBST and incubated with goat anti-rabbit or mouse-IgG secondary antibodies (peroxidase conjugated, ZSGB-BIO, Beijing, China) at room temperature for 1 h. The immunoreactive blots were developed [39 (link)].

Blood inside organs were removed by rinsing tissues with PBS. Tissues were snap-frozen under liquid nitrogen. Tissues were homogenized in RIPA buffer (Sigma) with protease (Roche), deacetylase (nicotinamide, Tricostatin A) and phosphatase inhibitors (Roche)^{7 (link)}. Protein concentrations of samples were determined by Lowry assay and each amount of protein (30–50 ug per sample) were loaded for SDS-PAGE. Antibodies from the following companies were used for Western blot analysis: Phospho-H2Ax (1:500, NBP1-19255-Novus Biological), PAR (1:500, AM80100UG-Millipore), actin (1:5000, sc-8432-Santa Cruz), nitrotyrosine (1:500, 06-284-Millipore) acetyl-lysine (1:1000, 9441-Cell signaling), HIF1a (1:400, 14179-Cell Signaling), GLUT1 (1:500, ab652-Abcam), PDK4 (1:1000, 3820-Cell Signaling), LDHA (1:1000, 3582-Cell Signaling), SDHA (1:10000, ab14715-Abcam), Ndufs4 (1:1000, ab87399-Abcam,), SOD2 (1:1000, ab16956-Abcam) SOD2-Ac (1:1000, ab137037-Abcam), GDH (1:1000, 12793-Cell Signaling). Blots were blocked in 5% BSA-TBST. Antibodies were diluted in 5% BSA-TBST. Protein bands were visualized with chemiluminescence assay (Pierce) with secondary antibodies coupled with HRP. The protein abundance was analyzed by densitometry with ImageJ. SDHA or actin were used as a loading control for quantification.

Protein extracts were prepared using lysis buffer for Western blot. A BCA protein assay kit (Beyotime, Shanghai, China) was implemented to detect the protein concentration. Equal amounts of the supernatant protein (50 μg) were separately subjected to 10% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). Primary antibodies were incubated overnight at 4 °C with polyclonal antibodies against VEGFR2, TEM1, TEM8, and β-actin. Antibodies against TEM1 (sc-377221; 1:250) and β-actin (sc-8432; 1:1000) were gained from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). Antibodies against TEM8 (ab21269; 1:250) and VEGFR2 (ab5473; 1:250) were purchased from Abcam (Cambridge, UK). After hybridization with a horseradish peroxidase-conjugated secondary antibody, blots were visualized using a chemiluminescence detection kit (Beyotime, Shanghai, China).

Proteins were extracted using urea buffer and analyzed by Western blotting as previously described in Pickard et al. (19 (link)). Primary antibodies used were mouse mAb to BiP (1:1000; sc-376768; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit pAb to collagen-I (1:500; OARA02579; Gentaur, Kampenhout, Belgium), mouse mAb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; clone GAPDH-71.1; MilliporeSigma), mouse mAb to vinculin (1:800; V9131; MilliporeSigma), and mouse mAb to β-actin (1:2000; sc-8432; Santa Cruz Biotechnology).

DADS, purchased from Fluka Co. (Milwaukee, Wisconsin, USA), was dissolved in Tween-80 and stored at −20°C after a 100-fold dilution with saline. The primary antibodies against cofilin1 (M1), p-cofilin1 (S3), LIMK1 (Q491) and p-LIMK1 (T508), Horseradish peroxidase (HRP)-conjugated secondary antibodies were provided by Abzoom (Dallas, TX, USA). The primary antibodies against Pak1 and Rock1 were purchased from Epitomics (Burlingame, CA, USA). The primary antibodies against destrin (ab11072), E-cadherin (ab40772), vimentin (ab92547), Ki-67 (ab66155), CD34 (ab81289), MMP-9 (ab38898) and TIMP-3 (ab39184) were provided by Abcam (Cambridge, MA, UK). Mouse monoclonal antibody against Rac1 (23A8) was purchased from Millipore (Billerica, MA, USA). The mouse monoclonal against β-actin antibody (sc-8432) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).