Peroxidase coupled secondary antibody

Whole-cell protein extracts were prepared as formerly described [37 (link)]. Primary antibodies against ENG (1:1000, #14606, Cell Signal, Danvers, MA, USA) and GAPDH (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) were combined with appropriate peroxidase-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and employed with an enhanced chemoluminescence system (Perkin Elmer, Waltham, MA, USA).

Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. For detection of proteins, membranes were incubated with antibodies diluted in 5% milk powder in Tris-buffered saline solution containing 0.1% Tween-20. Antibodies against phospho-groups were diluted in 5% BSA instead of milk powder. The following primary antibodies were used: goat anti-G2E3, mouse anti-hsc70, mouse anti-p53 (DO-1) (all Santa Cruz Biotechnology), rabbit anti-Caspase 3, rabbit anti-cleaved Caspase 3, mouse anti-Chk1, rabbit anti-PARP, rabbit anti-phospho-Chk1 (Ser317), rabbit anti-phospho-Chk2 (Thr68) (all Cell Signaling Technology), mouse anti-Chk2, mouse anti-Mdm2 (Ab-1), mouse anti-p21, mouse anti-PARP (all Calbiochem), mouse anti-actin (AC-15), rabbit anti-IgG (all Abcam), mouse anti-phospho-H2AX (Ser319) (Millipore), mouse anti-HA-tag (16B12, Covance), mouse anti-GFP (Clontech). Primary antibodies were detected by peroxidase-coupled secondary antibodies (Jackson ImmunoResearch Europe) using a Chemoluminescence Imaging System (Intas).

For direct lysis, cells were washed twice with PBS, and thereafter covered with 50 mM Tris-HCl, 100 mM DTT, 2% SDS, 0.1% bromophenol blue and 10% glycerol pH 6.8. Cell lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes, followed by a blocking step and incubation with primary antibodies as described previously [24 (link)]. To visualize protein bands, membranes were incubated with peroxidase-coupled secondary antibodies (Jackson Laboratories) for 1 h with subsequent detection using the SuperSignal West Pico Plus Chemiluminescent Substrate (34580, Thermo Scientific). Protein bands were quantified using ChemiDoc MP Imaging System using Image Lab Software. Following primary antibodies were used: rabbit anti-PECAM1 (LS-B4737, LSBio), mouse monoclonal antibody (mAb) against human lamin A+C (Chemicon, clone JoL2, mab3211, Abcam) for detection of human lamins (including human progerin), mouse monoclonal Lamin A/C antibody E-1 (Santa Cruz sc-376248) for detection of mouse and human lamins, and mouse mAb against α-tubulin (clone B-5-1-2, T5168, MilliporeSigma).

Cells were detached, washed with cold PBS, and centrifuged at 700 g for 10 min. The pellet was lysed in cold RIPA lysis buffer (20 mM TRIS pH 8, 150 mM NaCl, 1% Triton-X 100, 0.1% SDS, 1% sodium deoxycholate) containing protease and phosphatase inhibitors for 30 min on ice. Lysates were centrifuged at 10 000 g for 30 min at 4°C to remove debris.
Protein concentration of cell extracts was determined by the Bradford method (Bio-Rad, 5000006). Lysates were resolved by SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were blocked with 10% nonfat dry milk in Tris-buffered saline containing 0.01% Tween-100 for 1 h at room temperature (RT) and then incubated with specific primary antibodies used at final concentration of 1 μg/ml. The antibodies used are as follows: rabbit polyclonal anti-NOX4 (Santa Cruz Biotechnology, sc-30141), rabbit polyclonal anti-NOX1 (Abcam, ab55831), rabbit monoclonal anti-Nrf2 (D1Z9C) (Cell Signaling Technology, Euroclone, Pero (MI) Italy, 12721S), mouse monoclonal antitubulin (Sigma-Aldrich, T5168), and mouse monoclonal anti-Lamin A/C (Sigma-Aldrich, L1293). Immunocomplexes were detected through peroxidase-coupled secondary antibodies (Jackson), followed by enhanced chemiluminescence (GE Healthcare Life Sciences). Densitometry was done using Quantity One 1-D Analysis software (Bio-Rad).

FAS antibody (Jo-2) was purchased from Becton Dickinson. Antibody against the MET cytoplasmic domain was purchased from Life Technology (3D4/37-0100). Antibody against the C-terminal domain of human MET (L41G3), the MET phosphorylated tyrosine (Y1234/1235)(#3126), the phospho-ERK (Thr202/Tyr204)(#9106), phospho-AKT (Ser-473) (#9271), MCU, and cleaved Casp3 (asp175, #9661) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against cytochrome C(20E8) was purchased from BD Biosciences (San Jose, CA). Antibody against calnexin (ab75801) was purchased from Abcam (Cambridge, MA), antibody against FACL4/ACSL4 (NBP2-16401) from Novus Biologicals (Oakville, ON, Canada), antibody against GFP from Sigma (Saint Louis, MO). Green-fluorescent Alexa fluor 488 conjugated anti-mouse IgG and red-fluorescent Alexa fluor 594 conjugated anti-rabbit IgG were purchased from Invitrogen. Antibodies against PARP-1 (sc-7150), GAPDH (sc-32233, ERK2 (sc-154) and AKT (sc-8312) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-coupled secondary antibodies were from Jackson Immunoresearch Laboratories (West-Grove, PA).

HEK cells were transfected with Nfasc155 constructs for 24 h, then the cells were washed in PBS and solubilized on ice for 15 min in 1% Triton™ X-100, 140 mM NaCl, 20 mM Tris-HCl, pH 7.4 containing protease inhibitors. Proteins (50 µg) were loaded on 7.5% SDS-PAGE gels, transferred, and immunoblotted with a mouse antibody against Myc (1:2000) or a mouse antibody against α-tubulin (1:2000; MABT205; Merck). Immunoreactivity was revealed using peroxidase-coupled secondary antibodies (1:5000; Jackson ImmunoResearch) and BM chemiluminescence kit (Roche). The integrated densities of each protein band were measured with ImageLab software (Bio-Rad).

Whole cell lysates were prepared with RIPA lysis buffer (1% Triton X; 1% Desoxycholate, 0,1% SDS; 150 mM NaCl; 10 mM EDTA; 20 mM Tris-HCl; pH 7.5; 100.000KIE Trasylol) freshly supplemented with 2M Urea, Pefablock, 1μg/ml Leupeptin / Aprotinin, 1μg/ml Pepstatin A and 1μM Microcystin. Protein concentration was measured using the BCA protein assay (Thermo Fisher Scientific) and adjusted to equal amounts for all samples. For immunoblotting, protein lysates were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, blocked with 5% milk / TBS Tween and incubated with the following antibodies, each diluted in 5% BSA in Tris-buffered saline containing 0,1% Tween20: PARP (C-2-10; Calbiochem), Caspase 3 (8G10), cleaved Caspase 3 (5A1E), Bcl-xL (54H6), Akt, Mcl1 (D35A5), Bcl-2 (50E3), Bax, Bim (C34C5), and LC3a/b (all Cell Signaling Technology). Bcl-xL (H-5) and HSC70 (B-6) (both Santa Cruz), beta Actin (rabbit polyclonal), Noxa (EPR9735B) and Puma (EP512Y, all Abcam). Primary antibodies were detected with peroxidase-coupled secondary antibodies (Jackson). All immunoblot analyses were performed in triplicate to ensure the reproducibility of the results.