Liteablot turbo

A RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% TRITON X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) was used to prepare cell lysates. Proteins were quantified, separated in a 12% SDS polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane. After incubation in blocking solution, 5% BSA in TBS-T (Tris-Buffered Saline: 0,02 M Tris, 0,136 M NaCl, 0,001 % Tween-20, pH 7.6), the membrane was incubated overnight with a primary antibody at 4 °C. Antibodies used were rabbit polyclonal antibody against Syndecan4 (ABT157, Merck Millipore, Burlington, VT, USA) dilution 1:250, and goat polyclonal antibody against β-actin (#J1805, Santa Cruz Biotechnology, Dallas, TX, USA), dilution 1:1000. The membrane was washed with TBS-T and incubated for 1 h with the secondary antibody. Band visualization was carried out by the chemiluminescence system LiteAblot TURBO (Euro Clone, Pero, Italy). The relative intensities of the protein bands were analyzed with ImageJ software. β-actin levels were used as controls for protein loading.

Standard techniques were employed for SDS-PAGE and Western blotting to PVDF membranes using the antibody COQ6 12481-1-AP (Proteintech). Peroxidase-conjugated anti-rabbit IgG was used as secondary antibodies (Santa Cruz). Visualization of antibody protein complexes was achieved by enhanced chemiluminescence (LiteAblot Turbo, EuroClone) and the ChemiDoc™ XRS+ System (Bio-Rad).

Inflamed and non-inflamed biopsies, obtained from the colon of IBD patients, were frozen, homogenized and then lysed using a lysis buffer composed of Tris-HCl 10 mM pH 7.4, EDTA 100 mM, NaCl 100 mM, SDS 0.1%, and protease inhibitor cocktail 1% (Sigma, Saint Louis, MO, USA). The protein concentration was determined using the Bradford reagent (Sigma, Saint Louis, MO, USA). Samples were run in a denaturing 10% polyacrylamide gel (Thermo Fisher Scientific, Carlsbad, CA, USA) and were transferred to a PVDF membrane (Thermo Fisher Scientific, Carlsbad, CA, USA) that was incubated overnight at 4 °C with primary antibodies (anti-actin 1:10000 (42 kDa), anti-MMP9 1:1000 (92 kDa), and anti-MMP2 1:1000 (74 kDa); Sigma Saint Louis, MO, USA). The secondary antibodies were an anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (Cell Signaling, Danvers, MA, USA), diluted 1:40000 and an anti-rabbit HRP-conjugated secondary antibody (OriGene, Herford, Germany) diluted 1:1000. The reaction was developed with a chemiluminescence reagent containing luminol (Euroclone, Milan, Italy). Chemiluminescence was developed using LiteAblot^® TURBO (Euroclone, Milan, Italy) and exposed on Kodak Biomax film. MMP protein expression was quantified using the ImageJ software, version 1.45s and was reported as percentage with respect to actin.

After each treatment, cells were washed with phosphate-buffered saline and lysed in fresh ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.5, 500 µM EDTA, 100 µM EGTA, 1.0% Triton X-100 and 1% sodium deoxycholate) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma Aldrich). For immunoblot analysis, an equal amount of proteins (30 µg) was resolved in SDS-polyacrylamide gels (8–12 %) and transferred onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA) as previously described [25 (link)]. Thereafter, membranes were incubated with primary antibodies appropriately diluted. All primary antibodies were diluted in 1× Tween Tris-buffered saline with 5% bovine serum albumin or 5% non-fat dry milk according to the manufacturer’s protocol and incubated over night at 4 °C. All Santa Cruz Biotechnology primary antibodies were diluted 0.2 µg/ml, Cell Signaling Technology and MERCK antibodies were diluted 1:1000. Proteins were revealed by enhanced chemiluminescence (LiteAblot® TURBO; EuroClone). Densitometric analysis of the bands was performed using Image J Software v1.51 (NIH, Bethesda, MD, USA) using GAPDH for normalization.

MKN45 and MKN74 cells were lysed and proteins (50 μg) were separated by 10% SDS- polyacrylamide gels, followed by electro-transfer onto a nitrocellulose membrane. For EGFR and Erk1/2 western blotting only MKN45 cells were lysed and proteins (50 μg) were separated by 8% and 12% SDS-polyacrylamide gels respectively, followed by electro-transfer onto a nitrocellulose membrane. The membranes were sequentially incubated with blocking buffer (TBS-Tween containing 5% nonfat dry milk or 5% BSA respectively) for 1 hour at room temperature, and then overnight at 4°C with one of the following antibodies: anti-TGR5 (1:2000, #ab72608 Abcam), anti-FXR (1:2000 #sc-13063 Santa Cruz Biotechnology), anti-EGFR (1:1000, #2232 Cell Signaling), and anti-phospho-EGFR antibody (1:1000, #4407 Cell Signaling), anti-Erk1/2 (P44/42 MAPK) (1:1000, #46955 Cell Signaling), anti-phospho-Erk1/2 (p-P44/42 MAPK) (1:1000, #91015 Cell Signaling). Primary antibody were detected with the horseradish peroxidase (HRP)-labeled secondary antibodies (1:10000, BioRad) for 1 hour at room temperature. After washing with TBST, protein bands were visualized by Lite Ablot TURBO (Euroclone) according to the manufacturer's instructions.

Cells were lysed in lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, Milan, Italy). Lysates were separated on 6% SDS polyacrylamide gel and transferred onto Hybond-C extra membranes (GE Healthcare, Chicago, IL, USA). Membrane was blocked overnight at 4 °C with 5% milk and 3% BSA. Then, membrane was incubated 1 h at room temperature with mouse anti-human Rb (1:300, Santa Cruz Biotechnologies, Dallas, TX, USA) antibody (Ab) and overnight with mouse anti-β-actin Ab (1:3000, Santa Cruz Biotechnologies), followed by the incubation (room temperature, 1 h) with HRP-conjugated anti-mouse secondary Ab (Cell Signaling Technology, Milan, Italy). Peroxidase activity was visualized with the LiteAblot® PLUS and LiteAblot® TURBO (EuroClone, Milan, Italy) kits and densitometric analysis. The expression of Rb was evaluated both in Rb WT and KD MCF-7 cells.

5637 and T24 cells, untreated or treated CPS (300 μM) for different times, were lysed in a lysis-buffer containing protease inhibitor cocktail (Sigma Aldrich). Lysates were separated on sodium dodecyl sulphate polyacrylamide gel (9 and 14%) and transferred. After blocking with 5% low-fat dry milk in phosphate-buffered saline (PBS) 0.1% Tween 20 for l h, blots were incubated with the primary Abs: anti-LC3, anti-p62, anti-AMPK, anti-pAMPK, anti-Atg4C, or anti-GAPDH, followed by the appropriate HRP-conjugated antibodies. To assess the autophagic flux, cells were treated with CPS alone or in combination with BAF (25 nM). The LC3 conversion was also assessed in BC cells treated for 72 h with CPS (300 μM) alone or in combination with NAC (10 mM). In addition, western blot was performed in lysates from siGLO or siBeclin 1 BC cells, treated or not with CPS (300 μM) for 72 h, using anti-Beclin 1 or anti-LC3 Abs, to evaluate silencing efficacy and autophagy respectively. Moreover LC3 levels were investigated in lysates from siGLO or siPTCH2 BC cells treated, as above described, with CPS. The detection was performed using LiteAblot Turbo (EuroClone, Milano, Italy) and densitometric analysis was carried out evaluating three independent experiments by Chemi-Doc using Quantity One software (Bio-Rad, CA, USA). GAPDH levels were used as loading control.