Image labtm software

Proteins were extracted from primary cultured cortical neurons as described^{16 (link)}. Protein extracts (10 µg) were subjected to SDS-PAGE and blotted onto nitrocellulose membranes, which were treated first with anti-OPA1 (1/500, BD Biosciences, Le Pont de Claix, France) and anti-GAPDH (1/500, Merck Millipore MAB374, Molsheim, France) primary antibodies, and then with horseradish peroxidase-conjugated secondary antibodies (1/5000, Abcam ab6789 and ab6721, Paris, France) as described^{21 (link)}. After enhanced chemiluminescent detection, signals were analyzed using ChemiDoc^TM MP Imaging System and quantified by Image LabTM Software (Biorad®, Marnes la Coquette, France).

Total soluble leaf proteins were extracted from C, D, CR and DR plants according to Petersen and Bock [36 (link)], and resolved by electrophoresis on either 10 or 15% SDS-PAGE. Gels were blotted onto nitrocellulose membranes (Hybond^TM-ECL^TM, Amersham Biosciences, UK) at constant voltage (100 V), for 90 min. Membranes were incubated in blocking buffer (TBS containing 0.1% v/v Tween and 5% w/v defatted milk), for 1 h at room temperature. Membranes were challenged with primary antibodies (PsbP6 AS06 167; PsbQ AS06 142-16; GS2 AS08 296; FNR AS10 1625; RA AS10 700; RbcL AS03 037; ATP synthase β AS05 085; CSP41b AS08 298; light harvesting complex Lhcb1 AS09 522; elongation factor EF1α AS10 934 - Agrisera, Sweden), which were diluted in blocking buffer according to manufacturer’s instructions, and used at 4 °C overnight. A HRP-conjugated anti-rabbit antibody (1:60,000 dilution, GE Healthcare, UK) was applied for 1 h, at room temperature. Detection was carried out with ECL (GE Healthcare) following manufacturer’s instructions. Chemiluminescent signals were measured using a ChemiDoc^TM XRS+ (Bio-Rad) and images were analysed by using the Image Lab^TM Software (Bio-Rad). The protein accumulation levels are the mean of three biological samples presented with SD values. Significant differences were analysed by Student’s t test (^*P ≤ 0.05, ^**P ≤ 0.01).

Lung samples were homogenized with a tissue lyser (Qiagen, Hilden, Germany), proteins were isolated using the NucleoSpin® RNA/Protein Kit (Macherey-Nagel, Düren, Germany) and protein concentration was measured with a protein quantification assay (Macherey-Nagel).
20 µg proteins per lane were loaded, fractionated by SDS-PAGE (polyacrylamide gel concentrations in Table 1) and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked (blocking conditions in Table 1), incubated with the primary antibody, washed, incubated with the secondary antibody (antibody details in Table 1), washed and developed using the Clarity Max™ Western ECL Blotting Substrate (Bio-Rad) and the ChemiDocTM Touch Imager (Bio-Rad). Protein bands intensities were assessed with the Image LabTM Software (Bio-Rad), normalized according to the loading control and expressed as percentage of the CD mean of the respective membrane.

Western blot analysis was performed as described previously [26 ]. Western blot was imaged using a chemiluminescent horseradish peroxidase substrate (Millipore, Bedford, USA) on a Gel Doc^TM XR+ System with Image Lab^TM Software (Bio-Rad, USA). The antibodies used in this study were as follows: CCNB1 antibody (55,004-1-AP, 1:500, Proteintech, Rosemont, USA), CCND2 antibody (10,934-1-AP, 1:500, Proteintech), PCNA antibody (10,205-2-AP, 1:2000, Proteintech), and β-actin antibody (6008-1-Ig, 1:5000, Proteintech), and two secondary antibodies (ab150113 and ab150077, 1:5000, Abcam, Cambridge, UK). Quantification of the western blot was performed using the Image Lab (Bio-Rad).

Vehicle or TQ-treated breast cancer cell lines and tumor tissues obtained from the mouse study were lysed in RIPA lysis buffer (Li et al., 2010 (link)). Nuclear extracts of TQ treated or control cells were prepared using TransAM nuclear extract kit according to the manufacturer’s instructions. Whole cell lysates was resolved on a 10% SDS polyacrylamide gel electrophoresis (SDS–PAGE) gel. The proteins were electro-transferred to a nitrocellulose membrane (Bio-Rad), blocked with Blocking One (Nacalai Tesque, Inc.), and probed with antibodies of interest overnight at 4°C. The blot was washed with tris-buffered saline with 0.1% Tween-20, exposed to HRP-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence using Western Bright Sirius HRP substrate (Advansta). Images were captured using a ChemiDoc XRS+ imaging system and analyzed using Image Lab^TM software (Bio-Rad, Hercules, CA, United States) as described previously (Chua et al., 2010 (link)). Densitometric analysis was done using ImageJ software.

Proteins were separated using 12 or 4–15% Tris-glycine gels (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (Millipore) using semi-dry transfer (Bio-Rad). The blots were incubated for 1 h at room temperature in 5% (w/v) non-fat dry milk (Bio-Rad) in TBST buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween20) followed by the overnight incubation at 4°C with primary antibody. After 1 h incubation at room temperature with a 1:10,000 dilution of HRP-conjugated anti-mouse or anti-rabbit IgG F(ab)₂ (GE Healthcare Life Sciences, Piscataway, NJ), blots were developed using ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Sciences, Piscataway, NJ) (3 (link)). The protein bands were quantitated with ChemiDoc Imager and Image Lab^TM software (Bio-Rad, Hercules, CA).