Novex ap chromogenic substrate

Cells were seeded on Petri dishes (100 mm) and cultured to reach 90% confluence. Then, they were treated with experimental medium for 24 h. The protein isolation and western blots were conducted in accordance with previous study [16 (link)]. Primary antibodies used in the study were: anti-SOD1 (1:1000, Cell Signaling Technology, Leiden, WZ, The Netherlands, #71G8), anti-cleaved PARP1 (1:1000, Cell Signaling Technology, Leiden, WZ, The Netherlands, #D64E10), anti-GAPDH (1:2000, SantaCruz Biotechnology, Inc., Dallas, TX, USA, sc-59540). Novex^® AP Chromogenic Substrate (Thermo Fisher Scientific Inc, Waltham, MA, USA) was used to visualize bands. Quantification of bands intensity was measured with Image J software. The results are expressed as a relative expression normalized to control value (1.000).

Cells were lysed on ice with RIPA protein extraction buffer containing protease inhibitor cocktail (Sigma-Aldrich). Proteins (60 μg) were resolved on 10% SDS-PAGE and were transferred on PVDF membranes. The membranes were blocked for 1 h in 5% non-fat milk and incubated with a primary antibody for 18 h at 4°C. The antibodies used were goat polyclonal anti-WWOX (cat. no. sc-20529), mouse monoclonal anti-ARK1 (cat. no. sc-56881), goat polyclonal anti-KLF8 (cat. no. sc-69294), rabbit polyclonal anti-JAK1 (cat. no. sc-277) (all from Santa Cruz Biotechnology). Subsequently, the membranes were washed with TBST and incubated with the appropriate secondary antibody conjugated with alkaline phosphatase (Sigma-Aldrich) for 1 h at room temperature (RT). Next, the membranes were washed with TBST and developed with Novex^® AP Chromogenic Substrate (Invitrogen). GAPDH was used as a reference protein. The relative protein amount was assessed with ImageJ (NIH) based on the integrated density of the bands.

Transfected HEK293T cells were detached, centrifuged, and washed in ice-cold DPBS. Cell pellets were lysed in RIPA buffer (Sigma) containing 1% v/v protease inhibitor cocktail (Sigma). The protein concentration of clarified lysates was determined using a BCA assay (Thermo Fisher). Lysates were denatured, and 20 μg were loaded into SDS PAGE gels. Proteins were transferred onto nitrocellulose membranes (Thermo Fisher) and probed for EBNA1 (Merck, clone 1H4, monoclonal, cat MABF2800, dilution 1:1000) and a loading control, Vinculin (Sigma, clone hVIN-1, monoclonal, cat V9264, dilution 1:5000). Secondary antibodies (Life Technologies, Goat anti-Rat IgG A18868, polyclonal, and Abcam Rabbit Anti-Mouse IgG H&L (Alkaline Phosphatase) ab6729, dilutions 1:10000) were detected using the Novex™ AP Chromogenic Substrate (Invitrogen). Image J⁴⁸ was used for densitometry analysis. To obtain a normalisation factor specific to each lane, the Vinculin signal for each lane was normalised against the lane with the strongest signal. This was then multiplied by the EBNA1 signal of the same lane to get a normalised EBNA1 experimental signal. Finally, to control for inter-blot variability, normalised EBNA1 values were divided once more against the average normalised signal of each lane present within the blot (except the negative control).

SDS-PAGE was performed using a 12% polyacrylamide gel. Five microliters of total protein extract (approximately 10 µg) was loaded on the gel, and 5 µL of CLEARLY Stained Protein Ladder (Takara Bio) was used as a protein molecular weight marker. After electrophoresis, the proteins were transferred to a nitrocellulose membrane with a Tris-glycine-methanol buffer. The membrane was blocked for 1 h in Tris buffered saline supplemented with 5% (w/v) skim milk, 1% (w/v) bovine serum albumin and 0.6% (v/v) Tween 20. A monoclonal antibody against mCherry (Milipore) was used at a 1:1000 dilution to probe the membrane at 4 °C, overnight. Alkaline phosphatase conjugated secondary antibody (Vector) was used at a 1:1000 dilution for 1 h at room temperature, and the proteins exposed with Novex AP Chromogenic substrate (Invitrogen), according to the manufacturer’s protocol.

Immunoblot analysis of protein was performed as described in Bougdour et al. (2013) (link). Briefly, ~10⁷ cells were lysed in 50 μL lysis buffer (10 mM Tris-HCl, pH6.8, 0.5% SDS [v/v], 10% glycerol [v/v], 1 mM EDTA and protease inhibitors cocktail) and sonicated. The protein extracts were separated by SDS-PAGE, and transferred to a PolyVinyliDene Fluoride membrane (PVDF; immobilon-P, Millipore) by liquid transfer. M were then blocked with PBS buffer containing 0.01% Tween 20 (v/v) and 5% nonfat dry milk. Appropriate primary antibodies diluted in PBS containing 0.03% Tween 20 (v/v) were used to probe the membrane. Primary antibodies were detected using alkaline phosphatase or horseradish peroxidase conjugated secondary antibodies with Novex AP Chromogenic Substrate (Invitrogen) or Supersignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific), respectively.

Cells were lysed using RIPA buffer with protease and phosphatase inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined with the Bradford method (Bio-Rad Laboratories). SDS-PAGE was followed by semi-dry transfer (45 min, 250 mA, Fast Blot, Biometra) of the proteins to a PVDF membrane (Sigma-Aldrich). The process was supported by Whatman® Lens Cleaning Tissue (Sigma Aldrich), the use of which requires pre-soaking in TB 10 × transfer buffer. Protein transfer was confirmed by transient staining with Ponceau red. Precluding of non-specific binding of the antibodies was accomplished via membrane blocking with non-fat milk (5% solution) in TBST buffer 1 × .
Anti-WWOX/AP-2γ/AP-2α (Thermo-Fisher; catalogue numbers: PA5-29701, PA5-49862 and MA5-14856, respectively) were added as primary antibodies at a dilution of 1:1000 to 1% non-fat milk in TBST buffer solution. Overnight incubation was followed by three times washing using TBST buffer. Following this, the PVDF membranes were incubated with secondary anti-rabbit antibodies conjugated with alkaline phosphatase (1:30,000) and the analyzed protein were visualized with Novex® AP Chromogenic Substrate (Invitrogen).
Anti-GAPDH antibody was used as reference (sc-59540, Santa Cruz Biotechnology Inc.). The relative amount of protein was determined by densitometric analysis with ImageJ software.