Supersignal west pico reagent

Snap-frozen mouse brain cortices were homogenized on ice in RIPA buffer containing complete protease inhibitors (Roche Diagnostics Cat# 06538304001, Mannheim, Germany). The homogenates were rotated for 30 min at 4 °C to ensure extraction of membrane proteins. After centrifugation at 15000 Å ~ g for 120 min, supernatants were collected and protein concentrations were measured with the bicinchoninic acid protein assay reagent (Thermo Scientific, Grand Island, NY, USA). Equal amounts of protein samples were resolved on 4–12% NuPage Bis-Tris gels purchased from Invitrogen. Following incubation with the indicated primary antibody, an appropriate horseradish peroxidase-conjugated secondary antibody was added. Immunoreactivity was detected by chemiluminescence using Super Signal West PICO reagent (Thermo Scientific). Image-J software was used to quantify the mean gray value for a fixed area of each protein band [27 (link)]. The original full-blot images can be found in “Additional File_Full gel blots”.

Cells were lysed in SDS-PAGE loading buffer containing 1% SDS. Cell lysates were cleared by brief sonication using a microsonicator (UR-20P, Tomy Seiko, Japan) and subsequent centrifugation. Cleared lysates were subjected to SDS-PAGE followed by electrotransfer to a PVDF membrane according to standard procedures. Proteins of interest were reacted with corresponding primary antibodies and detected by a chemiluminescence method using a secondary HRP-conjugated antibody and a Super Signal West Pico reagent (Thermo Fisher Scientific, USA). Chemiluminescence was detected using a ChemiDoc XRS Plus imaging system (Bio-Rad, USA) and quantified using ImageLab software (Version 2.0, Bio-Rad).

An in vitro prenylation assay was used to measure defective protein prenylation (19 (link), 36 (link), 37 (link)). Briefly, uRab proteins in cell lysates were prenylated by incubation with recombinant Rab geranylgeranyltransferase and a synthetic biotin-isoprenoid lipid substrate, and detected on PVDF membranes using streptavidin-680RD (LI-COR) (37 (link)).
Some lysates were also analyzed by Western blotting for the presence of uRap1A using anti-Rap1A (Santa Cruz Biotechnology, sc-1482) with anti-goat 680RD secondary antibody (LI-COR) (30 (link), 37 (link)). β-Actin (Cell Signaling Technology, 3700) or a narrow doublet of endogenous biotinylated protein approximately 73 kDa (often appearing as a broad singlet) was used as a sample loading control (37 (link)). Blots were scanned on a LI‑COR Odyssey imager and analyzed using Image Studio v5.2.5. MK protein was detected in liver homogenates by Western blotting using 1 μg/mL rabbit polyclonal antibody (Antibodies Online) against amino acids 179–228 of human MK, and HRP-conjugated anti-rabbit IgG. Blots were developed using SuperSignal West Pico reagent (Thermo Fisher Scientific) and scanned on a Fusion FX7 imaging system (Etablissements Vilber Lourmat SAS). Densitometry was performed using ImageJ v2.0.0 (NIH).

Analysis of protein abundance was performed by Western blot, according to standard techniques. Cell lysates were collected using RIPA buffer with protease inhibitors, and were quantified using BCA assay. Some cell lysates were collected using 2× Laemmli buffer directly. Samples were boiled for 5 min, separated by 10% SDS PAGE, and transferred to PVDF membranes by semi-dry transfer. Blots were probed using AhR (BML-SA-210, Enzo Life Sciences, Farmingdale, NY, USA) and GAPDH primary antibodies (sc-365062, Santa Cruz Biotechnology, Dallas, TX, USA). Chemiluminescence signal was developed using horse radish peroxidase conjugated secondary antibodies (SouthernBiotech, Birmingham, AL, USA) and SuperSignal West Pico reagent (Thermo Fisher Scientific, Waltham, MA, USA). Images were captured using a G:BOX imaging system and GeneSys software version 1.5.9 (Syngene, Cambridge, UK).

Total cellular proteins were extracted in lysis buffer (containing 1% NP40, 20 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L EDTA, 10% glycerol, protease, and phosphatase inhibitor tablets). Protein extracts (30 µg) were run on 4–12% NuPAGE® Novex® Bis-Tris gels (Life Technologies) and transferred at 4 °C onto Immobilon polyvinyldifluoride membranes (Thermo Scientific). After blocking, membranes were incubated at 4 °C overnight with primary antibodies specific for: caspase-3 (#9662), cleaved caspase-3 (Asp175) (#9661), Myosin Light Chain 2 (MLC2) (#3672), phospho-MLC2 (Ser19) (#3675), LC3 A/B (#4108), and p-(S)-CDKs Substrate (#9477), obtained from Cell Signaling Technology. Antibodies against GAPDH (#MAB374) were purchased from Millipore. Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit (Southern Biotechnology) antibodies were then incubated for 1 h and revealed with the SuperSignal West Pico® reagent (Thermo Fisher Scientific) or the ECL^TM Prime Western Blotting Detection System (GE Healthcare) using an ImageQuant LAS 4000 software-assisted imager (GE Healthcare).

Cell suspension (1 × 10⁷ cells/ml in HBS) was exposed to nsPEFs, immediately diluted 5-fold into pre-warmed HBS, and incubated at 37 °C for the appropriate time periods. Cells were collected by centrifugation and then snap-frozen in liquid nitrogen. Cells were lysed in SDS–PAGE loading buffer containing 1% SDS and then sonicated using a microsonicator (Model UR-20P, Tomy Seiko, Tokyo, Japan). Cell lysates were cleared by brief centrifugation and in turn subjected to SDS-polyacrylamide gel electrophoresis followed by western blot analysis as described previously^{10 (link)}. Antigen–antibody complexes were reacted with an HRP-conjugated secondary antibody and then incubated in Super Signal West Pico reagent (Thermo Fisher Scientific). Chemiluminescence was detected using ChemiDoc XRS Plus analyzer (BioRad).