Gradient gel

Cells growing in 6-well plates were treated with trypsin (Sigma-Aldrich), washed with PBS and sedimented by centrifugation. Next, the cell pellets were lysed using RIPA lysis buffer supplemented with protease inhibitors (Sigma-Aldrich) and incubated on ice for 30 min. The lysates were centrifuged at 17000 × g for 30 min at 4 °C, and the supernatant was collected. The protein concentration was measured using the Bradford method. Lysates were mixed with Bolt LDS Sample Buffer (Life Technologies) supplemented with β-mercaptoethanol (final concentration 5%). Samples containing equal amounts of protein (20 μg) were separated by SDS-PAGE on gradient gels (4–12%, from Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad Laboratories). After blocking in Odyssey Blocking Buffer diluted 1:1 in TBS for 1 hour, the membranes were incubated with primary antibodies at 4 °C overnight. After washing with TBS-T, the blots were incubated with secondary antibodies for 1 hour at room temperature. The blots were visualized using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA). Drp1 was detected using mouse antibody (BD Bioscience) at a 1:2000 dilution. Rabbit anti-GAPDH (Abcam) was used at 1:50000 dilution. Fluorescently labeled secondary antibodies (Li-Cor Biosciences) were used at 1:20000 dilution.

Patients were selected from those that were admitted to Istanbul University, Cerrahpasa School of Medicine, Department of Clinical Microbiology and Infectious Diseases in Istanbul. Experiments were performed in accordance with the established institutional guidelines and approved protocols from the local ethics committee (Name: Istanbul University-Cerrahpasa, Ethics Committee for Clinical Research, Date and Number: 17 January 2017, 22142), and after obtaining informed consent from all participants. Patients’ characteristics are shown in Table 1. Isolation of mononuclear and polymorphonuclear leukocytes from patients’ peripheral blood was performed as described above. Isolated leukocytes were subsequently lysed in standard Laemmli buffer containing β-mercaptoethanol by boiling at 95°C for 10 min. Lysates were cleared upon centrifugation at 11,000g for 10 min. SUMO conjugates were separated on 4–12% gradient gels (Invitrogen) or homemade 8% SDS–PAGE for further analysis by Western blot. Importantly, equal amounts (20 μg) of total protein were loaded in each well to allow fair comparison of SUMO profiles between different samples, that is, HIV(−) or HIV(+) leukoctye extracts.

Samples for western blot analysis were harvested as described (Räsänen et al., 2008). Samples were run on 4–12% gradient gels (Life Technologies, Carlsbad, CA, USA). Using Trans‐Blot Turbo system, proteins were transferred to nitrocellulose membrane (both from Bio‐Rad, Hercules, CA, USA) and blocked with 5% (w/v) nonfat powdered milk in TBS (20 mm Tris/HCl pH 7.5, 150 mm NaCl, and 0.1% Tween‐20). Immunoreactive proteins were visualized with appropriate primary and secondary antibodies using ECL detection (Bio‐Rad).
The following primary antibodies were used according to the manufacturers’ recommended dilutions: rabbit monoclonal anti‐phospho‐p44/42 MAPK (ERK1/2, Thr202/Tyr204), rabbit polyclonal anti‐p44/42 MAPK (ERK1/2), rabbit monoclonal anti‐phospho‐MEK1/2 (Ser217/221), mouse monoclonal anti‐MEK1/2 (L38C12), rabbit polyclonal anti‐phospho‐STAT3 (Y705), mouse monoclonal anti‐STAT3 (124H6), and rabbit monoclonal anti‐ATF4 (D4B8) (all from Cell Signaling Technology, Danvers, MA, USA). Rabbit polyclonal anti‐GAPDH was from Sigma‐Aldrich. The secondary antibodies used in western blotting were affinity‐purified horseradish peroxidase‐coupled anti‐rabbit IgG H+L and anti‐mouse IgG H+L (both from Jackson ImmunoResearch, Suffolk, UK).

Cells were lysed in a RIPA buffer (300 mM NaCl, 10 mM TrisHCl pH 8.0, 10 mM KCl 1 mM EDTA, 1% Nonidet-P40, 1% Na Deoxycholate, 0.1% SDS, 1 mM PMSF) and protease inhibitors cocktail (11697498001, Roche, Indianapolis, IN, USA), sonicated on ice with a Branson 450 sonicator through two cycles of ten 2 s pulses (1 min pause between cycles), and then cleared by centrifugation at 13,000× g for 10 min. Equal amounts of protein were separated by 4–12% gradient gels (Life Technologies, Great Island, NY, USA) and transferred to nitrocellulose membranes (10401396, Whatman, Maidstone, UK). Membranes were blocked with 5% milk in TBST (150 mM NaCl, 10 mM Tris HCl pH 7.5, 0.1% Tween 20) for one hour at room temperature. Immunostaining was accomplished by overnight incubation with primary antibodies (Table S5) followed by one hour of incubation with dye-conjugated secondary antibodies according to manufacturer’s instructions. Protein detection was achieved by using an Infrared Odyssey system (LiCor, Lincoln, NE, USA). Densitometric analysis of Western blots was performed by using ImageJ software. Actin was used as normalization probe.

NRVCMs were lysed 72 h post viral transduction by three freeze–thaw cycles in RIPA buffer (20 mM Tris, 10 mM DTT, 500 mM sodium chloride, 1% NP40, 12,5% glycerol) supplemented with phosphatase and protease inhibitor cocktails (Roche, Germany). The cell lysate was then centrifuged at 12,000 × g for 20 min. to remove cell debris, and the supernatant was used for protein concentration measurement by DC-assay (Bio-Rad Laboratories). Proteins prepared as above were resolved on 10% SDS–PAGE, or commercially available 4–12% gradient gels (Life Technologies), and transferred to a polyvinylidenefluoride membrane, blocked for 2 h in 5% dry-milk prepared in 0.1% TBST at room temperature (RT), followed by the incubation with primary antibodies overnight at 4 °C, 4× washes with 0.1% TBST and final incubation with a suitable HRP-coupled secondary antibody (1:10,000) (Santa Cruz, Germany). Protein signals were developed using ECL-select chemiluminescence kit (GE Healthcare) and visualized on Fluorchem Q imaging system (Biozym). Quantitative densitometry was performed with the help of ImageJ/Fiji version 1.46. All the uncropped immunoblot images have been included as Supplementary Fig. 7. All source data underlying the graphs presented in the main figures using immunoblot densitometry data are made available as Supplementary Data 1.