Western lightning ecl pro

Nitrocellulose (Hybond ECL, GE Healthcare, Chicago, IL) was pre-wetted with PBS-T (PBS containing 0.1% Tween 20) and then spotted under vacuum with 50 µl 10% seminal plasma, 10% blood plasma, or 20 µg/ml semen amyloids (SEVI or SEM1(86–107) fibrils) or the corresponding monomeric peptides. Wells were then washed once with PBS-T under vacuum. The nitrocellulose membrane was then immediately transferred to a blocking solution (PBS-T containing 5% non-fat dry milk and 1% BSA) and incubated at 4°C with gentle shaking for 2 hr. The membrane was then washed three times for 5 min before incubation with anti-Amyloid Fibrils OC Antibody (EMD Millipore, Billerica, MA) or Anti-Amyloid Oligomers A11 antibody (Abcam, Cambridge, United Kingdom) (both used at 1:2500 in PBS-T with 1% BSA). Primary antibody incubation was allowed to proceed overnight at 4°C with gentle shaking. The membrane was then washed three times for 5 min each before addition of HRP-conjugated rabbit IgG (GE Healthcare) (used at 1:5000 in PBS-T with 1% BSA). Secondary antibody incubation was allowed to proceed for 2 hr at 4°C with gentle shaking. The membrane was then washed three times for 10 min each, and then developed by 1 min incubation with the Western Lightning ECL Pro (Perkin Elmer, Waltham, MA), and exposed using chemiluminescence film (Amersham Hyperfilm ECL, GE Healthcare).

Western blot analysis was performed as described previously [34 (link)]. HepG2 cells were grown in 100 mm culture dishes with or without METF. Cell extracts were prepared by lysing the cells in RIPA buffer. 30 μg of protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membranes (Protran®, Whatman® Schleicher & Schuell). The blots were then blocked and incubated with specific primary antibodies, followed by incubation with anti-species-specific secondary antibodies. To confirm equal protein loading per sample, we used anti-calnexin or anti-GAPDH antibody. Finally, detection of specific proteins was performed by enhanced chemiluminescence reagent (Western Lightning ECL Pro, PerkinElmer). Quantitative measurement of immunoreactive band intensities was performed by densitometric analysis using the Scion Image software (Scion Corporation, Frederick, MD, USA). Data were then converted into fold changes (FC) of the control.

Western blot analysis was performed on TCA (trichloroacetic acid) extracts prepared from culture volumes corresponding to 10 OD₆₀₀ units. Protein extracts were precipitated and pellets were resuspended in loading buffer. After boiling, samples were loaded on 7.5% polyacrylamide gels. GFP tagged proteins (Rnr3, Rnr2, Rnr4, Rad53) were detected with mouse α-GFP antibody (Roche 11814460001, 1:5000). Rnr1 was detected with rabbit α-Rnr1 antibody (Agrisera AS16 3639, 1:10000). Rabbit α-tubulin antibody was used for the detection of tubulin, our loading control (Abcam ab184970, 1:20000). Secondary antibodies used were goat α-Mouse IgG H&L (Abcam Ab6789, 1:10000) and α-Rabbit IgG (Cell Signalling #7074, 1:5000). Secondary antibodies were detected using Western Lightning ECL Pro (PerkinElmer).

Immunoblotting and immunoprecipitation were performed essentially as described previously^{39 (link)}. To analyze TM2D3 by immunoblotting, samples were denatured at 50 °C for 5 min before loading to the gels. The first antibodies on the membrane filters were visualized with secondary antibodies conjugated with horseradish peroxidase, a Western Lightning ECL Pro (PerkinElmer) chemiluminescent substrate, and a LAS-1000plus (Fujifilm) or ChemiDoc Touch (Bio-Rad) luminescent image analyzer; or secondary antibodies conjugated with near infrared dyes and Odyssey CLx (LI-COR).

HepG2 cells were grown in 100 mm culture dishes with or without LC combined or not with F. Cellular extracts were obtained lysing the cells in RIPA buffer as previously described [24 (link)]. Thirty micrograms of proteins were separated by SDS–polyacrylamide gel electrophoreses (SDS-PAGE) and electrophoretically transferred to nitrocellulose membranes (Potran^®, Whatman^® Schleicher & Schuell). The blots were then blocked and probed with specific primary antibodies, followed by incubation with anti-species-specific secondary antibodies.
To confirm equal protein loading per sample, we used antibody anti-calnexin or anti-GAPDH. Detection of specific proteins was performed by enhanced chemioluminescence reagent (Western Lightning ECL Pro, Perkin Elmer). Quantitative measurement of immunoreactive band intensities was performed by densitometry analysis using the Scion Image software (Scion Corporation, Frederick, MD, USA). Only for AMPK inhibitor experiments and CaMKII, immunoreactive bands were visualized by Uvitec Alliance LD9 gel imaging system (Uvitec, Cambridge, UK).
Data were then presented as ratio between treated cells and control.

After SDS‐PAGE, the gels were processed for immunoblotting using Trans‐Blot Turbo Transfer System (BioRad, Hercules, California, USA) and 0.2 μm pore‐size nitrocellulose membranes (BioRad). Immunoblot detection was done with Western Lightning ECL Pro (Perkin Elmer, Winter St. Waltham, Massachusetts, USA), and a ChemiDoc XRS + system (BioRad) or exposure to Amersham Hyperfilm ECL (GE Healthcare).