Complete ultra protease inhibitor cocktail

Advillin-CreERT2:Bdnf ^fl/fl mice were injected with tamoxifen or vehicle (corn oil) as described above, in the section Tamoxifen-induced recombination. Two weeks after the final injection, spinal cords (5.0 mm of lumbar enlargement) and TGs were homogenized in 20× volume lysis buffer (pH 7.0) containing Tris-HCl (100 mM), NaCl (1.0 M), EDTA-Na₂ (4 mM), bovine serum albumin (2%), Triton X-100 (2%), sodium azide (0.1%), and complete ULTRA protease inhibitor cocktail (Roche). Homogenates were centrifuged at 14 k × g for 30 min at 4°C. Spinal cord supernatants were diluted 10× and TG supernatants diluted 5×. All supernatants were immediately assayed with the Human BDNF SimpleStep ELISA kit (Abcam). Absorbance was measured at 450 nm with a Biotek H4 Plate Reader.

Vero-TMPRSS2 cells were infected with either WT or S mutants of SARS-CoV-2 at an MOI of 0.1. After 24 h, infected cells were lysed in lysis buffer (1% NP-40; 20 mM Tris-HCl, pH 7.5; 150 mM NaCl; and 5 mM EDTA) supplemented with cOmplete ULTRA protease inhibitor cocktail (Roche Diagnostics). Proteins in each lysate were resolved by SDS-PAGE and transferred onto Immobilon-P PVDF membranes (Merck). The blots were incubated with the following primary antibodies: anti-SARS-CoV-2 N or anti-SARS-CoV-2 S (GTX632269, GTX632604, GeneTex). HRP-conjugated anti-β-actin antibody (PM053-7, MBL) was used to detect the loading control. Immune complexes were detected using HRP-conjugated secondary antibodies and the Immobilon Western Chemiluminescent HRP Substrate (Merck).

For each lane, 1x10⁸ cells were isolated and lysed on ice for 30 mins in 100 μL of lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.25% NP-40) supplemented with one tablet of cOmplete ULTRA protease inhibitor cocktail (Roche). Note: As the cutaneous Leishmania donovani strain expressed plasmid encoded proteins to a lower level than the wildtype strain 1S2D, 5X more lysate was used as input in cutaneous Leishmania co-IP experiments. Lysate was clarified by centrifugation at 21,000x g for 20 mins. 10 μL of clarified lysate was mixed with SDS Sample Buffer and used as ‘Input’ lanes. 1 μL of FLAG or HA antibody was added to each tube of clarified lysate and the reaction volume adjusted to 500 μL with Lysis Buffer. Lysate:Antibody mixture were incubated for 2h at RT with gentle agitation on a tube revolver. For each tube, 25 μL of Protein A/G Magnetic beads (Pierce) were washed in Lysis Buffer for 5 mins and added to the mixture. Tubes were incubated for an additional hour at RT on a tube revolver. Beads were isolated from the tubes using a DynaMag2 magnetic stand (Life Technologies). The beads were then washed three times with 1 mL of Lysis Buffer and resuspended fully each time. Captured and bait proteins were eluted off the beads by incubating in 2X SDS Sample Buffer for 10 mins followed by SDS-PAGE.

Cell aggregates were resuspended in RIPA (radioimmunoprecipitation assay) buffer (Sigma) containing cOmplete Ultra Protease Inhibitor Cocktail (Roche), sonicated (3 × 10 s) on ice to ensure complete lysis (Bioruptor Plus, Diagenode, Liège, Belgium), and centrifuged for 13,000g for 10 min at 4 °C to remove debris and undigested cells. Protein concentration was quantified using a BCA protein assay kit (Thermofisher). Fifty-microgram protein was loaded on a 10–12% SDS-PAGE gel. Samples were transferred to a nitrocellulose membrane and detected using primary antibodies listed in Additional file 1: Table S2.

Expi293F cells (Thermo Fisher Scientific) were transfected with an expression plasmid for full-length human CHCHD2 c-terminally tagged with a PA tag48 (link) according to the manufacturer's protocol. The cells from a 50 ml culture were lysed via sonication in lysis buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 0.1% Triton-X and cOmplete Ultra protease inhibitor cocktail (Roche), followed by centrifugation at 27,216 g for 15 min. The supernatant (10 ml in total) was incubated with 100 μl of anti-PA tag NZ-1-Sepharose for 1 h at 4 °C, and the beads were sequentially washed with 1 ml of wash buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, followed by 1 ml of interaction buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol and 0.2% n-decyl-β-D-maltopyranoside (DM) (Anatrace, D322). The CHCHD2-bound beads were incubated in the presence or absence of 306 μg of purified bovine heart complex IV (provided by Dr Tsukihara) for 1.5 h at 4 °C and washed twice with 1 ml of the interaction buffer. Co-precipitated proteins were eluted with SDS-containing buffer and analysed via SDS–PAGE and subsequent Coomassie Brilliant Blue (CBB) staining or western blotting with anti-complex IV subunit IV (Abcam, 20E8C12).