Rabbit anti gapdh

We examined the expression levels of different genes using western blot analysis, as recently shown by our group [69 (link)]. Briefly, SKBR3 and ZR75 (1 × 10⁶ cells) were seeded and treated with the chalcone compounds DK-13 or DK-14 at 10 μM and 20 μM concentrations for 48 h. Cell lysates were harvested, and an equivalent protein concentration of 30 μg was separated on gradient polyacrylamide gels (PAGE) and blotted onto nitrocellulose membranes followed by probing with the following primary antibodies: anti-mouse Bax (Thermo Fisher Scientific, Mississauga, ON, Canada), anti-mouse Bcl-2, anti-rabbit Pro-caspase-3, anti-rabbit anti-ERK1/ERK2 antibody, anti-rabbit phosphorylated ERK1/ERK2 and anti-rabbit JNK1/JNK2/JNK3 (Abcam, Cambridge, MA, USA). To ensure equal protein loading in all samples, re-probing of membranes was undertaken with the housekeeping protein anti-rabbit GAPDH (Abcam, Cambridge, MA, USA).
Different protein bands were developed by chemiluminescence ECL-Western blotting (Pierce Biotechnology, Waltham, MA, USA) as described by the manufacturer. For relative quantification of protein expressions, ImageJ software was used to analyze the acquired western blotting images. Bands’ intensities normalized to β-actin was used to determine the relative protein expression in each cell line.

Subcellular fractionation of drug-treated cultures were performed essentially according to ref. 65 (link). Fractions were analysed by 10% TGX SDS-PAGE and proteins blotted onto nitrocellulose membranes using TransBlot turbo (Bio-Rad). Blots were incubated with anti-TH (rabbit; Thermo Scientific), anti-GM130 (mouse; BD Transduction Laboratories), anti-GAPDH (rabbit, Abcam) or anti-porin (mouse; Abcam) primary antibodies. Horse radish peroxidase coupled to anti-rabbit or anti-mouse antibodies, ChemiDoc and Image Lab software (Bio-Rad) were used for chemiluminiscence visualization and analysis of blots. The intensity of target proteins was standardized with the corresponding loading control for each subcellular fraction. Treated samples were compared to untreated, which were normalized to a value of 1. The sample size in all cases was n = 3. Results are given as mean value ± SD and a two-way comparison using the t-test was performed in order to determine statistical significance.

Cultured cells were washed with PBS and lysed with Mammalian Protein Extraction Reagent (M-PER, Thermo Scientific) with the phosphatase inhibitor cocktail (PhosSTOP, Roche) and the protease inhibitor cocktail (cOmplete, Roche). Protein samples were separated on a 4-15% SDS-PAGE gel (TGX gel, Bio-Rad) and transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were incubated in Block Ace (BioRad) at 4°C overnight and then with anti-NCL-Hamlet DYSF (mouse, 1:2,000, Leica), anti-RFP (DsRED, rabbit, 1:1,000, Thermo Scientific) and anti-GAPDH (rabbit, 1:5,000, Abcam) as primary monoclonal antibodies. Membranes were washed three times in PBS with 0.1% Tween-20, then with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5,000, Cell Signaling Technology) or anti-mouse IgG (1:10,000, GE Healthcare Life Science) as secondary antibodies. All antibody incubations were performed at room temperature (RT) for 1-hr. Blots were developed using Immun–Star™ WesternC™ Chemiluminescence Kit (BioRad) and imaged using an A600 imager (Azure Biosystems). Band intensities were analyzed using Fiji (Fiji Is Just ImageJ, (Schindelin et al., 2012 (link))) image processing software.

Lysates and immunoprecipitates were analyzed by SDS-PAGE on mini-PROTEAN Stain-free Precast gels (Biorad, Marne la coquette, France), transferred to nitrocellulose membrane, and immunoblotted with primary antibodies, including anti-FLAG M2 (Sigma, Saint Quentin Falavier, France), anti-myc (9B11, Cell Signaling, Leiden, Holland), polyclonal anti-protein C (hpc4, Cell signaling), rabbit anti-GAPDH (Abcam, Cambridge, UK), polyclonal anti-GFP (gift from S. Miserey-Lenkei, Curie Institute; Paris, France), and polyclonal anti-KLHL21 (Invitrogen, Paris, France) antibodies. Thereafter, the membranes were incubated with a horseradish peroxidase-conjugated mouse or rabbit secondary antibody (1:5000 dilution). The images were obtained with chemiluminescence (Clarity Max Western EXL, Biorad; Marne la coquette, France) using a luminescent image analyzer (ChemiDoc XRS+, Biorad, Marne la coquette, France) and quantified with ImageLab software version 8.1.0 (Biorad, Marne la coquette, France).

Tissue and cell samples were lysed in RIPA buffer containing a protease inhibitor cocktail. Protein concentration in the lysed sample was detected using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, USA). After that, samples containing 5× load buffer were heated for 5 min at 95°C. Equal amounts of protein were separated by 4-20% SDS-PAGE, and transferred to PVDF membranes (BIO-RAD, USA). Then, the membranes were blocked with 5% non-fat milk in TBS with 0.1% Tween-20 for 1 h and incubated with rabbit anti-GAPDH (Abcam, 1:10000), rabbit anti-lysozyme (Abcam,1:1000) overnight, respectively. After the washing, goat anti-rabbit secondary antibodies (Bioss, 1:1500) were used to incubate the membranes. Finally, the optical protein bands were developed using efficient chemiluminescence (ECL) kit, and light emission was captured using the Versa DOC 4000 imaging system.