Fluor s max multiimager

HT29 cells were seeded in 25 cm² flasks at a density of 2 × 10⁴ cells/cm², and after 24 h, they were treated with 5 μM of compound 5. After 24 h, both treated and control cells were washed twice with PBS and lysed in RIPA buffer [50 mMTris/HCl, 150 mMNaCl, SDS (1% v/v), Triton X-100 (1% v/v), 1 mM EDTA, pH 7.6]. Cell lysates were centrifuged at 12,000× g for 20 min, and the protein concentration was determined by using the Bio-Rad protein assay method (Bio-Rad, Hercules, CA, USA). The proteins were detected resolving samples on a 12% gel in Tris-Glycine buffer at 200 V for 50 min. Western Blot was performed in transfer buffer at 100 V for 1 h. The nitrocellulose membrane was incubated with primary antibodies for rabbit anti-p21 (Millipore, Billerica, MA, USA) or rabbit anti-p27 (Millipore, Billerica, MA) or mouse anti-Bax (Millipore, Billerica, MA, USA) or mouse anti-α tubulin (Invitrogen, Walthman, MA, USA) for 1 h. Detection of immunoreactive bands was performed by using a rabbit or mouse HRP-conjugated secondary antibody (GE Healthcare, Milan, Italy), followed by Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Milan, Italy). Densitometry analysis of immunoreactive bands was done by Fluor-S Max MultiImager (Bio-Rad, Hercules, CA, USA). Relative quantification of bands was performed by using an α-tubulin signal as control.

SDS-PAGE using 10% or 12% gels was used to separate the proteins. Western blotting was subsequently performed by blotting the separated proteins onto an Immobilon-P polyvinylidene fluoride membrane (Merck Japan) using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). After blotting, the membrane was blocked in 5% nonfat milk (FUJIFILM Wako Pure Chemical) in TBST (pH 7.6) and incubated in a rat anti-PA tag antibody (1:10,000; FUJIFILM Wako Pure Chemical). Other primary antibodies used included the anti-DENV E antibody (1:3000; GeneTex, Irvine, CA, USA) or the mouse anti-DENV prM antibody (1:3000; GeneTex). After incubating membranes with the primary antibodies and washing three times with TBST, the membranes were incubated for 1 h in horseradish peroxidase (HRP)-conjugated anti-rat IgG antibody (1:10,0000; FUJIFILM Wako Pure Chemical). Immobilon Western Chemiluminescent HRP substrate (Merck Japan) was used for the detection of protein bands. Membranes were imaged using a Fluor-S MAX Multi-Imager (Bio-Rad).

Proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide gels and were subsequently subjected to western blotting. After SDS-PAGE, the proteins were blotted onto a polyvinylidene fluoride (PVDF) membrane with a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). For western blotting, after blocking with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBST, pH 7.6), the membrane was incubated in either a 1,000-fold dilution of a rabbit polyclonal antibody against MGAT2 (hGnT II) (GeneTex, Los Angeles, CA, USA) or a 1,000-fold dilution of rabbit IgG B4GALT1 (hGalT I) (EnoGene Biotech, New York, NY, USA). The membrane was subsequently washed with TBST and then was incubated for 1 h in a 1:10,000 dilution of either an anti-mouse or anti-rabbit IgG antibody labeled with horseradish peroxidase (GE Healthcare Japan, Tokyo, Japan). Detection was performed by using ECL Plus western blotting reagent (GE Healthcare Japan). Specific bands were detected on a Fluor-S MAX MultiImager (Bio-Rad). For the lectin blots, the PVDF membrane was incubated with 2 μg/ml FITC-conjugated RCA120 (J-OIL MILLS, Tokyo, Japan), then washed with TBST. Specific fluorescent bands were detected with a Molecular Imager FX system (Bio-Rad).

HT29 cells were treated with 49 µM of (R)-1 for 24 h. Cell were lysed in according to ref. 23. In brief, the cells were lysed for 20 min in an HEPES buffer and the protein concentration was determined by using the Bio-Rad protein assay method (Bio-Rad, Hercules, CA, USA). The proteins were resolved on a 10% density gel and immunoblotted with p21 (Cell Signaling Technology, Danvers, MA#2946, USA) or Myc (Cell Signaling Technology, Danvers, MA #5605, USA) or β-actin (Cell Signaling Technology, Danvers, MA #4967, USA) antibodies. The nitrocellulose membrane was incubated with secondary horseradish peroxidase-conjugated antibodies (GE Healthcare, Chicago, IL #NA931V or #NA9340, USA), developed as described in the previous section, and the quantification was done by Fluor-S Max MultiImager (Bio-Rad) using the β-actin signal as the control.

Control, siRNA, and scramble HeLa cells were washed twice with PBS and lysed for 1 h in lysis buffer (HEPES, pH 7.4 40 mM, glycerophosphate 60 mM, p-nitrophenylphosphate 20 mM, Na₃PO₄ 0.5 mM NaCl 250 mM, Triton X-100 1%, PMSF 0.5 mM, and 10 mg/mL each of aprotinin, leupeptin, pepstatin and antipain, Sigma-Aldrich) at 0°C. Cell lysates were centrifuged at 12,000 × g for 20 min. Supernatants were collected and protein concentration determined by using the Bio-Rad protein assay method (Bio-Rad, Hercules, CA, United States). The proteins were resolved on a 7.5% polyacrylamide gel and immunoblotted with a rabbit anti-human α5 integrin subunit (1:1000 in PBS Tween, Cell Signaling Technology, Danvers, MA, United States), or a rabbit anti- β-actin (1:2000 in PBS Tween, Sigma-Aldrich) antibodies. Detection of immunoreactive bands was performed by using a HRP-conjugated secondary antibody (1:20,000 in PBS Tween, GE Healthcare, Milan, Italy) followed by WESTAR EtaC 2.0 (Cyanagen, Bologna, Italy). Densitometry analysis of immunoreactive bands was done by Fluor-S Max MultiImager (Bio-Rad). Relative quantification of α5 integrin subunit was performed by using β-actin signal as control.