Anti β actin

As read out for TGF-β signaling activity, protein expression of phosphorylated Smad2 was measured by Western blot. Hs27a cells were grown as monolayer and treated with ZOL (10 - 500 μM) for 48 hours. Subsequently, 500,000 SCP2 cells were plated in 6 wells plates and incubated for 24 hours. Pure active TGF-β1 (5 ng/mL) or harvested supernatant of Hs27a cells was added to the confluent SCP2 cells. After 1 hour incubation, the supernatant was removed. Total cell lysates were size fractionated and transferred to a membrane as described previously [24 (link)].
Membranes were exposed to primary antibodies (anti-pSmad2; Cell Signaling Technology, anti-β-actin; MP Biomedicals). Binding of antibodies was determined using horseradish peroxidase (HRP)-conjugated secondary antibodies (DAKO) and visualized with Lumi-light^plus (Roche Diagnostics). Band density was evaluated by ImageJ software.

Immunoblot, immunoprecipitation and subcellular fractionation were performed as described previously [15 (link)]. For immunoprecipitation assay, PIP5K1α or VEGFR2 antibody was used to pull down the immune-complex, with an IgG antibody (BD Biosciences, San Jose, CA, USA) used as negative control. Various antibodies were used in immunoblot as shown below: PIP5K1α (Proteintech Inc. and Cell Signaling Technology), Phospho-S473 AKT, CDK1, cyclin D1 (Cell Signaling Technology and Santa Cruz Biotechnology), VEGF, VEGFR1, VEGFR2, p27, Cyclin A2, anti-GAPDH (Santa Cruz Biotechnology), ERα (Biosite), Ki-67 (DAKO), MMP-9 (Abcam), anti β-Actin (MP Biochemicals), cyclin E (Upstate Inc.). Immunoblot analysis was performed and semi-quantified using ImageJ Image Analysis Software (NIH, MD, USA).

Gamitrinib conjugated with triphenylphosphonium was prepared as described previously [33 (link)]. MitoTracker, Fura-2-AM, and tetramethylrhodamine methyl ester (TMRM) were purchased from Molecular Probes, Ryanodine was from Santa Cruz Biotechnology. Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP) was from Calbiochem. 1,2-bis(o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid acetoxymethyl ester (BAPTA), cyclosporine A (CsA), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), tetracaine, and thapsigargin (Thap), and N-acetylcysteine (NAC) and all other chemicals, were from Sigma.
Anti-CEBP homologous protein (CHOP) antibodies were obtained from Cell Signaling; anti-RyR, anti-IP₃R, anti-eIF2α and anti-cytochrome c antibodies from Santa Cruz Biotechnology; anti-cyclopholin D from Calbiochem; anti-eIF2α[pS52] from Invitrogen; anti-β-actin from MP Biomedicals; and anti-TRAP1 from BD Biosciences.

Western blots were carried out for ID4 and BRCA1 protein. Seventy two hours after transfection with miR-342 precursor or negative control, MDA-MB-231, HCC1937 and MCF7 cells were collected by trypsinization and resuspended in 300 ul of 1X SDS sample buffer (62.5 Mm Tris-HCl pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT), supplemented with protease inhibitors cocktail (Calbiochem).
Protein quantification was performed using the BCA protein assay (Thermo Scientific). Forty micrograms of protein extract were loaded on 7% to 15% polyacrylamide gels and transferred to nitrocellulose membrane. The following primary antibodies were used: anti-ID4 (82-12, dilution 1∶1000, Calbioreagents), anti-BRCA1 (dilution 1∶800, Cell Signaling Technology), anti-β-Actin (dilution 1∶5000, MP Biomedicals), anti-Vinculin (dilution 1∶5000, Sigma-Aldrich) and anti-α-Tubulin (dilution 1∶5000, Sigma-Aldrich). Signals were detected using enhanced chemiluminescence system (Thermo Scientific).
Protein level quantification was performed using the software Imagelab©, by subtracting the global background of each membrane from the volume of intensity of the antibody detection bands (rectangle encompassing the whole band in the volume tools). The level of a protein in a certain sample was measured as the ratio between the quantifications of the band for the specific-antibody and the band for loading control.

Cells/embryos were lysed in 2x sample buffer (0.125 M Tris pH 6.8/4% SDS/20% glycin/5% beta-mercaptoethanol/0.025% bromphenol blue). Proteins were separated by SDS-PAGE and transferred to Immobilon-P Transfer membranes (Millipore) by wet tank blotting. Blots were blocked in 5% nonfat dried milk powder (AppliChem) in PBS/0.1% Tween 20. Primary antibodies: anti-HA HRP (rat monoclonal; clone 3F10, Roche; HRP, horseradish peroxidase-conjugated; 1:5,000–1:10,000), anti-Flag HRP (mouse monoclonal; clone M2, Sigma), anti-GFP HRP (mouse monoclonal; MACS molecular; 1:10,000), anti-DLL1 (1F9, rat monoclonal, [21 (link)] 1:1,000), anti-DLL4 (rabbit polyclonal against peptide C-GKIWRTDEQNDTLT; BioGenes; 1:50–1:100), anti-β-actin (mouse monoclonal; MP Biomedicals; 1:250,000–1:500,000), anti-β-tubulin I (Sigma; 1:500,000). Secondary antibodies: anti-mouse HRP, anti-rat HRP, anti-rabbit HRP (Amersham; 1:10,000). HRP was detected with ECL Western Blotting Detection Reagents (Amersham) with the Luminescent Image Analyser LAS-4000 (Fujifilm); signals were quantitated with ImageJ software.

Immunoblotting was performed as previously described [9 (link)]. In brief, whole protein lysates were separated by SDS/PAGE and transferred to nitrocellulose membranes (Whatman, Dassel, Germany). Membranes were incubated with the following primary antibodies diluted in 5% Milk/TBST-containing blocking solution overnight: anti-KPNA2 (rabbit polyclonal, 1:2000; abcam, Cambridge, UK), anti-stathmin (rabbit monoclonal, 1:1000; abcam), anti-E2F1 (rabbit polyclonal, 1:200; Santa Cruz, Heidelberg, Germany), anti-TFDP1 (rabbit polyclonal, 1:500; abcam), anti-ATF-2 (rabbit polyclonal, 1:200; Santa Cruz), anti-FBP-1/2 (goat polyclonal, 1:200; Santa Cruz), anti-c-JUN (rabbit monoclonal, 1:2000; Cell Signaling Technology, Frankfurt, Germany), anti-HMOX1 (rabbit monoclonal, 1:10,000; abcam), anti-GTSF1 (goat polyclonal, 1:200; Santa Cruz), anti-PARP (rabbit polyclonal, 1:500; Cell Signaling Technology), anti-β-tubulin (mouse monoclonal, 1:1000; Becton, Dickinson and Company, Franklin Lakes, USA) and anti-β-actin (mouse monoclonal, 1:10,000; MP Biomedicals, Illkirch, France). Blots were incubated with fluorescence-conjugated secondary antibodies (LI-COR Bioscience, Bad Homburg, Germany) for one hour and detection was performed using the Odysee Sa Infrared Imaging System (LI-COR Bioscience).