Chemidoc digital imaging system

Surface biotinylated material and total cell lysates were fractionated by gel electrophoresis on 4-20% Tris-glycine gels (Invitrogen, Waltham, MA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA). The S2 spike subunit was detected with mouse anti-S2 monoclonal 1A9 (GeneTex, Irvine, CA, USA, cat#GTX632604); cellular actin was probed for with anti-actin monoclonal sc-47778 (Santa Cruz Biotechnology, Dallas, TX, USA). Each experiment was repeated at least three independent times, and representative data or images are shown in the figures. Trichloroacetic acid (TCA)-precipitated pseudotyped particles were fractionated as described for biotinylated material. Protein was detected with specific antibodies directed against S2 and against VSV matrix (23H12; courtesy of Douglas Lyles; Kerafast) [42 (link)]. Immunoblots were visualized using HRP-conjugated mouse IgG or rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) secondary antibodies, and a ChemiDoc digital imaging system (Bio-Rad, Hercules, CA, USA). Immunoblot data were quantified using ImageLab software.

Briefly, the membranes were incubated in blocking buffer (5% skim milk in PBST (0.05% Tween-20 in PBS) for 1 h at room temperature and then probed using an anti-APP antibody 22C11 (APP 66–81) produced in house [45 (link)], anti-human SOD1 (Abcam, 1:10,000) and an anti-APLP2 95/11 a rabbit polyclonal antiserum raised against recombinant APLP2 (28–693) protein [46 (link)] and expressed in Pichia pastoris (as previously described) [47 (link)]. Loading control GAPDH was detected using Cell Signaling Technology antibody (#2118). Blots were incubated in primary antibody diluted in PBST overnight at 4°C. The next day, the membranes were washed in PBST buffer, incubated with a secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature and washed in TBST. Immunoreactivity was detected using the enhanced chemiluminescence reagent (ECL-plus, GE Healthcare, UK) and imaged on a ChemiDoc digital imaging system (BioRad, Australia). Protein expression levels were quantitated by densitometry analysis of band intensities using Image J/Fiji software (ver. 1.52e, NIH). The intensity value for each immune-reactive band was normalised to its corresponding housekeeping loading control to account for variability in protein loading across samples.

Cells were harvested with ice-cold PBS containing 10 mM EDTA. The protein contents of samples were measured by a BCA protein assay. Proteins were denatured at 98 °C for 10 min. Proteins (5 μg) were subjected to SDS-polyacrylamide gel electrophoresis (4–20% gradient polyacrylamide gels) and then electrophoretically transferred onto nitrocellulose membranes. Membranes were blocked with PBS/0.1% Tween 20 containing 5% nonfat dried milk at room temperature for 30 min. After washing with PBS/0.1% Tween 20, membranes were incubated with antibodies against MLKL (1:1000) or β-actin (1:10,000) in PBS/0.1% Tween 20 containing 5% bovine serum albumin at 4 °C overnight. Membranes were washed with PBS/0.1% Tween 20 and incubated with secondary antibodies conjugated with horseradish peroxidase in PBS/0.1% Tween 20 containing 5% nonfat dried milk at 4 °C for 1 h. After washing, proteins were visualized using enhanced chemiluminescence reagents. The quantification of chemiluminescent signals was performed with a ChemiDoc digital imaging system (Bio-Rad, Hercules, CA, USA).

Western blotting was performed as previously described (Sahin et al., 2017 (link)). Briefly, metathoracic legs were isolated and homogenized at 4°C in 40% RIPA, 60% lammeli+5% β-mercaptoethanol. The samples were spun for 10 min at 16,000×g and the supernatant was transferred to a new microcentrifuge tube. The supernatant was then boiled for 10 min, cooled on ice and then loaded onto 4–20% SDS-PAGE gels. Following electrophoresis, protein was transferred to blotting membrane, and blocked for 2 h at room temperature in superblock (Thermo Fisher Scientific) or 1% BSA in tris-buffered saline +0.01% Tween (TBST). Following primary antibody incubation and wash, blots were incubated with anti-peroxidase secondary antibodies (Jackson ImmunoResearch,1:10,000) for 2 h and washed three times in TBST. Blots were developed with SuperSignal West Pico Plus Chemiluminescence (Thermo Fisher Scientific) and imaged using a Chemidoc digital imaging system (Bio-Rad).

Cellular protein was extracted in lysis buffer containing 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1% Triton, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 50 mM β-glycerolphosphate, 2 mM Na₂VO₄ and protease inhibitors and cleared by centrifugation. The NE-PER fractionation kit (Pierce) was used for nuclear/cytoplasmic fractionation according to the manufacturer's instructions. Protein extracts were separated using SDS-PAGE, transferred to 0.45 uM Immobilon-P PVDF membrane (Millipore) and immuno-blotted using the indicated antibodies followed by ECL. The blots were analyzed using ChemiDoc digital imaging system (BioRad).