Nupage precast gel

Cells were freshly harvested and counted. 600,000 cells were lysed using lysis buffer containing 50% of TruPAGE LDS Sample Buffer (PCG3009; Merck) and 20% 2-mercaptoethanol. Cell lysates were heated at 98°C for 5 min, cooled down to RT, diluted 1:1 using water, and run in NuPAGE precast gels (NP0321BOX; Thermo Fisher Scientific). The gels were transferred to membranes using the iBlot system (IB401001; Thermo Fisher Scientific). The membranes were incubated with primary antibodies KCNQ3 (GTX54782, 1:1,000; GeneTex) and GAPDH (ab181602, 1:10,000; Abcam) at 4°C overnight, followed by IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (925-32211, 1:5,000; LI-COR). The membranes were visualised using the LI-COR Odyssey CLx system.

Whole cell extracts were prepared by lysis in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Na-deoxycolate, 0.1% SDS, 1% NP-40, 2 mM Na_2-EDTA), supplemented with protease inhibitors (Roche, Germany). Protein concentration was determined by Bio-Rad protein assay (Bio-Rad, CA, USA) [12 (link)] and 10–25 μg of proteins were separated on 4–12% Nu-PAGE pre-cast gels (Thermo Fisher Scientific). After blotting on PVDF and 1 h saturation in PBS containing 0.05% Tween-20 and 5% skim milk, membranes were incubated for 1 h or overnight with primary antibody, diluted in PBS containing 0.05% Tween-20 and 0.5% skim milk, washed three times for 10 min in PBS containing 0.05% Tween-20, incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (Bio-Rad) and the signals detected with Chemiglow by means of a FluorChem SP system (Alphainnotech, Germany). Primary antibodies were against: PARP, (BioMol, Germany, 1 μg/ml), Caspase 3 (9662, Cell Signaling, MA, USA, 1 μg/ml), Caspase 9 (9502, Cell Signaling, 1 μg/ml), LC3 (2775, Cell Signaling, 1 μg/ml), LC3B (D11 XP, Cell Signaling, 1 μg/ml), PINK1 (D8G3, Cell Signaling, 1 μg/ml), BAX (2D2 and N-20, Santa Cruz, 0.5 μg/ml), BAK (N-20, Santa Cruz, 0.5 μg/ml), cytochrome c (556432, Becton Dickinson, NJ, USA, 1 μg/ml). β-Tubulin (Sigma Aldrich, 1 μg/ml) was used as a loading control for cell extracts.

To confirm that the synthesized mRNAs could be translated into GFP or RBD proteins, 293FT cells were seeded at 3 × 10⁵ cells/mL in 6-well plates, grown for 24 h, and then transfected with 1 mg mRNA per well using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. To prevent secretion of proteins, cells were incubated with the protein transport inhibitor brefeldin A (5.0 μg/mL) for 8 h after transfection. The transfected cells were cultured at 37°C for 24 h and then collected and lysed using protein lysis buffer (Thermo Fisher Scientific, Cat #: 87787). Aliquots of lysate (20 μg protein) were resolved on 4–12% NuPAGE precast gels (Thermo Fisher Scientific) and transferred to PVDF membranes. RBD protein expression was analyzed using a rabbit polyclonal antibody SARS-CoV-2 Spike RBD Antibody (HRP) (Sino Biological, #40592-T62), which cross-reacts with SARS-CoV RBD protein. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was probed as a loading control.

Western blotting was performed as previously described^{51 (link)}. Briefly, uninfected and HIV-1 infected or untreated and Meth treated CD4⁺ T-cells (after incubation period) were collected in cell lysis buffer, protein lysates were separated on NuPAGE precast gels (Life Technologies Corp.), transferred to 0.45 μm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), and probed with appropriate primary antibodies followed by incubation with their respective secondary antibodies. Proteins were visualized with Western Lightning Plus ECL Substrate (PerkinElmer, Waltham, MA).
For immunoprecipitation assay, CD4⁺ T-cells were left untreated or treated with Meth (100 μM) and incubated for times indicated. Cells were lysed with cell lysis buffer (Cell Signaling Technology). Cell lysates were subjected to immunoprecipitation using Millipore PureProteome^TM Protein A and Protein G Magnetic beads, which were used according to manufacturer’s protocol (MilliporeSigma, Burlington, MA) and the immune-complexes were further processed by Western blotting^{51 (link)}.

iMDDCs were left untreated, or treated with cocaine in indicated concentration. After indicated incubation time, samples were collected in RIPA buffer. Protein lysates were separated on NuPAGE precast gels (Life Technologies Corp.), transferred to 0.45 μm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), and probed with appropriate primary antibodies followed by incubation with their respective secondary antibodies. Proteins were visualized with Western Lightning Plus ECL Substrate (PerkinElmer, Waltham, MA).
For immunoprecipitation assay, DCs were left untreated or treated with cocaine (1 μM) and incubated for times indicated. DCs were lysed with cell lysis buffer (Cell Signaling Technology). Immunoprecipitation was performed as previously described61 (link).