Trans blot turbo transfer apparatus

For Western blotting, 20–40 μg of each cell homogenate was separated via SDS-PAGE utilizing a 15% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo transfer apparatus (Bio-Rad). Membranes were blocked with 5% nonfat dried milk in TBS-0.1% Tween (TBS-T) for 20 minutes. Primary antibodies were diluted in TBS-T containing 10% Super Block T20 (Thermo Scientific, 37536) at appropriate dilutions (1:500–1:1000) and allowed to bind to membranes overnight at 4°C. Blots were washed (3x) for 10 min in TBS-T, the blot was then incubated for 1h at room temperature with a horseradish peroxidase conjugated secondary antibody at 1:5000 or an Alexa Fluor 488^® secondary antibody diluted in TBS-T containing 10% Super Block T20. Clarity Western ECL Substrate (Bio-Rad, 1705060) was used to detect the HRP of the secondary antibody. ChemiDoc MP imaging system and Image Lab software (Bio-Rad) were used to image and quantify blots. These experiments were conducted independently at least twice by utilizing 2–3 technical replicates in each experiment, and the images presented are representative samples.

Whole cell extracts were directly lysed into 2x Laemmli Sample buffer containing 5% β- mercaptoethanol and stored at −20°C until use. Total protein extracts were heat-treated at 95°C for 5 min, fractionated by SDS-PAGE and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer apparatus (BioRad, Hercules, California, USA). After incubation with 5% non-fat milk in Tris-buffered saline with 0.05% Tween (TBS-T) for 1 h, the membrane was incubated at 4°C overnight with polyclonal antibodies against MAGI2 (1:500) (ABCAM, Cat. No. ab97343) and β-Tubulin (1:1,000) (ABCAM, Cat. No. ab18207). Membranes were washed three times for 10 min and incubated with horseradish peroxidase-conjugated anti-mouse (1:2,000) or anti-rabbit (1:10,000) antibodies for 1 h at room temperature in 5% BSA in TBS-T (Jackson ImmunoResearch Laboratories, Inc.; Cat No. 111-035-003 and 115-035-003, respectively). Blots were washed with TBS-T three times and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, Cat No. 34094) on the ChemiDoc MP system (BioRad, Hercules, California, USA). The quantification of band intensities was done using the Image Lab 4.1 software and results were normalized to β-tubulin.

Cells were lysed with Protein Extraction Reagent (Fisher Scientific; 78510) supplemented with protease and phosphatase inhibitors (Fisher Scientific; 78442); then, the cell extracts were subjected to electrophoresis on 10% Mini-PROTEAN TGXTM Precast Gels (BIO-RAD; 4568033), and the separated proteins were transferred to a nitrocellulose membrane (BIO-RAD; 1704270) by using a Trans-Blot Turbo transfer apparatus (BIO-RAD). The membrane was incubated with 5% non-fat milk (BIO-RAD; 1706404) in PBST for 60 min at room temperature and with primary antibodies (Supplemental Table 1) overnight at 4 °C; then, the membrane was washed three times with TBST, incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000 dilution) for 30 min at room temperature, and washed three more times with TBST. Protein signals were developed with HRP-substrate (MILLIPORE; WBKLS0500) and images were acquired with a ChemiDocTM Imaging System (BIO-RAD).

To study the oxidative damage suffered by P. falciparum 3D7 across the intraerythrocytic cycle in HbAS RBCs, parasite proteins DNPH-derivatized (2.5 μg) were electrophoresed on 10% SDS–PAGE at 90 and 120 V for 150 min (Miniprotean tetracell, Biorad, Hercules, CA, USA). Gels were transferred 30 min on PVDF membranes employing a semi-dry Trans-Blot turbo transfer apparatus (Biorad, Hercules, CA, USA). PVDF membranes were blocked for 2 h at room temperature with 5% nonfat dry milk in PBS. Then, membranes were incubated with rabbit polyclonal anti-DNPH antibodies (Merck, St Louis, MO, USA) at 1:5000 in PBS-milk 5%, for 2 h at room temperature with gentle rocking, followed by incubation with peroxidase-linked anti-rabbit IgG antibody (Thermo Fischer Scientific, Burlington, ONT, Canada) at 1:5000 for 1 h at room temperature. Chemiluminescence signals were developed as was mentioned for the dot-blot assay.
Preparative SDS-PAGE, loaded with 50 μg by lane, were run at the same conditions and stained with Coomassie Blue Brilliant (CBB) following a general protocol [12 (link)]. Next, they were matched against oxyblots to select protein carbonylated bands.

Proteins were extracted using RIPA buffer containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Rockford, IL, USA) and 1 mM phenyl-methane-sulfonyl fluorid (PMSF). Proteins were transferred from the gel to the nitrocellulose membranes using a Trans Blot Turbo transfer apparatus (Biorad, Hercules, CA, USA). Primary antibodies for GFAP (1/4000, anti-mouse, Sigma-Aldrich, #G3893), NFATc4 (1/1000, anti-rabbit, Santa Cruz Biotechnology, #sc-13,036), β-Actin (1/10000, anti-rabbit, Cell Signaling Technology, #4970), vimentin (1/2000, anti-mouse, Sigma, #V5255), or CB1 (1/2000, anti-rabbit, Millipore, #07-069) were added to the membranes in 5% BSA TBS-T and were incubated overnight. Membranes were subjected to ECL (Biorad, Hercules, California, USA) detection on a LiCor Odyseey instrument (Lincoln, NE, USA).

Whole cell extracts were prepared with an alkaline lysis protocol as previously described.¹⁰⁰ (link) Briefly, three independent 1 ml overnight yeast cultures (biological replicates) were subcultured in 25 ml YEPD until mid-log phase (∼6 hours). 5 ml of culture were harvested before the addition of auxin, and every 15 min after addition of 1 mM IAA. Cells were resuspended in 0.5 ml 100 mM NaOH, and incubated at room temperature for 5 minutes. Cells were then harvested and resuspended in 75 μl LDS sample buffer (Pierce, 84788) supplemented with 1 mM DTT. Sample was heated to 70^oC and the extract separated from cell debris by centrifugation. 5-10 μl of cleared whole cell extract was run on a 4-12% Bis-Tris SDS-PAGE (Thermo, NP0323BOX) MOPS-SDS buffer at 200 V for 50 minutes. Proteins were transferred onto nitrocellulose membrane (Whatman Protran BA85, cat. No. 10401196) using the Trans Blot Turbo transfer apparatus (25 V, 1 A, 30 minutes) (Bio-Rad), and blocked with 5% milk. Blots were cut between the 75 kDa and 50 kDa marker bands. The top blot was probed with anti-FLAG M2 antibody (1:5,000 Sigma F1804), and the bottom blot was probed with anti-alpha tubulin [EPR13799] (1:5,000 Abcam ab184970). Secondary antibodies were used at 1:5,000 dilutions, and membranes visualized with ECL as described above.