Anti gapdh rabbit polyclonal antibody

Myotubes were trypsinized, pelleted and resuspended in 300 µL of Dulbecco’s phosphate buffer saline (D-PBS) depleted in Ca²⁺ and supplemented with 1-mM EGTA. Sonication of ice-cold cell suspension was performed with a Branson digital sonifier (amplitude 20%, duration 2 min, interval 5 s and pulse 5 s) and two successive centrifugations at 13,000 g for 1 min allowed to remove cell debris. 10 μg protein extracts were separated on 10% SDS-PAGE. Tank electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. AnxA5 (35 kDa) and AnxA6 (68 kDa) were, respectively detected with mouse anti-AnxA5 (Sigma, Saint-Louis, MI, USA) and anti-A6 (Santa Cruz Biotechnology, Dallas, TX, USA) monoclonal antibodies and GAPDH (loading control) was detected with a rabbit anti-GAPDH polyclonal antibody (Santa Cruz Biotechnology). Primary antibodies were diluted 1:1000 in saturation solution composed by Tris buffer saline (10-mM Tris, 150-mM NaCl, pH 8.0) supplemented with 0.1% Tween20 and 5% non-fat dry milk. Revelation was performed using secondary antibodies conjugated to horse-radish peroxidase (GE-Healthcare, Chicago, IL, USA) diluted 1:2000 in saturation solution and Opti-4CN™ colorimetric kit (Bio-Rad, Hercules, CA, USA). ImageJ software was used to measure the relative intensity of protein bands.

Cell extracts were prepared with RIPA buffer containing proteases and phosphatases inhibitors. For extracting cytoplasmic, nuclear, membrane and cytoskeletal proteins separately Compartimental Protein Extraction Kit (Merk Millipore, Darmstadt, Germany) was used according with manufacturer instruction. Proteins were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes and then treated with the following primary antibodies: anti-β-catenin mouse monoclonal (1:1000), anti-N-cadherin rabbit polyclonal (1:1000) (Santa Cruz Biotechnology), anti-E-cadherin mouse monoclonal (1:500) (Dako Corp.), anti-VEGF rabbit polyclonal (1:200) (Abcam, Inc.) antibodies. Anti-GAPDH rabbit polyclonal antibody (1:2000) (Santa Cruz Biotechnology) was used to normalize protein content. Horseradish peroxide-conjugated goat anti-mouse or goat anti-rabbit secondary antibody complexes were detected by chemiluminescence (Santa Cruz Biotechnology).

FM3A, MTT060562, and 3T3 cells, before and after treatment with transduction proteins, were used for western blotting. The samples were loaded onto a 5%–20% gradient polyacrylamide gel (Wako, Tokyo, Japan) and electrophoretically transferred to nitrocellulose membranes (GE Healthcare, Danbury, CT, USA). The membranes were blocked with 10% skim milk in PBS. The primary antibodies were anti-FLAG M2 mouse monoclonal antibody (Sigma Aldrich), anti-HA mouse monoclonal antibody (Sigma Aldrich), anti-MCM2 mouse monoclonal antibody (BD Biosciences), anti-DNA-PK_cs mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-DNA-PK S2056 (Mouse-S2053) rabbit polyclonal antibody (Assay Biotech, Sunnyvale, CA, USA), anti-P53 mouse monoclonal antibody (Merck, Darmstadt, Germany), anti-phospho-P53 (Ser 15) rabbit polyclonal antibody (Merck), anti-cleaved caspase-3 rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti-GFP mouse monoclonal antibody (Abcam), anti-PP2A rabbit polyclonal antibody (Cell Signaling Technology) and anti-GAPDH rabbit polyclonal antibody (Santa Cruz Biotechnology). The secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse IgG (GE Healthcare) and HRP-conjugated anti-rabbit IgG (GE Healthcare). Protein expression was detected using the Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA).

20 μg for each sample were run on a 10% SDS-PAGE and then transferred a polyvinylidene difluoride membrane (Pall Corporation). The membrane was blocked with 5% non fat dry milk in PBS containing 0.1% Tween 20 (PBST) for one hour and then probed with anti-FUT8 monoclonal antibody (Abcam, Cambridge, UK, 1:1000 dilution), or anti-GAPDH rabbit polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA, 1:1000 dilution). Detection was achieved using a secondary anti-rabbit HRP-conjugated IgG (Santa Cruz Biotech, Santa Cruz, CA, 1:1000 dilution). GAPDH was used as a control. Immunoreactive bands were visualised using ECL Western blotting kit (Amersham Biosciences, UK) and were normalized to those of GAPDH.

Samples of equal amounts were separated on SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Pall Corporation). The membrane was blocked with 5% non fat dry milk in PBS containing 0.1% Tween 20 (PBST) for one hour and then probed with anti-FUT4 monoclonal antibody (Abcam, Cambridge, UK, 1:1000 dilution), or anti-GAPDH rabbit polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA, 1:1000 dilution). Membrane proteins were detected by HRP-conjugated secondary antibody (Santa Cruz Biotech, Santa Cruz, CA, 1:1000 dilution). GAPDH was used as a control. Immunoreactive bands were visualised using ECL Western blotting kit (Amersham Biosciences, UK) and were normalized to those of GAPDH.