Ma1 21315

Cells were lysed in passive lysis buffer (Promega) and clarified by centrifugation. Cell lysates were mixed with Laemmli sample buffer containing 2.5% β-mercaptoethanol and heated at 90°C for 5 min before SDS-PAGE was performed. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and detected using primary rabbit polyclonal antibodies (pAbs) specific for IFI44 (Abcam ab172499), FKBP5 (Cell Signaling 8245), HA tag (Sigma-Aldrich H6908), FLAG tag (Sigma-Aldrich F7425), phospho-IKKα (Ser176)/IKKβ (Ser177) (Cell Signaling 2078), phospho-IKKε (p IKKε Thr501) (Rockland 600-401-267), and IRF-3 (Abcam ab25950) and mouse monoclonal antibodies (MAbs) against the His epitope tag (Thermo Fisher Scientific MA1-21315), the MYC epitope tag (Thermo Fisher Scientific 13–2500), phospho-IRF-3 (pIRF-3; Abcam ab76493), phospho-IkBα (pIkBα; Thermo Fisher Scientific MA5-15224), IkBα (Abcam ab32518), and actin (Sigma-Aldrich, A1978) followed by incubation with a 1:1,000 dilution of goat anti-rabbit (pAb) or anti-mouse (MAb) IgG antibodies conjugated to horseradish peroxidase (Sigma-Aldrich). Membranes were revealed by chemiluminescence using SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific), according to the manufacturer’s recommendations.

Proteins were mixed to reach a final concentration of 0.5 μM bovine TRiC and 5 μM PhLP2A fragment after the addition of either H₂O or ATP/AlFx (1 mM ATP, 1 mM AlNO₃, 6 mM NaF). Samples were separated on a 5% acrylamide gel containing 80 mM HEPES pH 8 and 1 mM MgCl₂. The running buffer consisted of 80 mM HEPES pH 8, 1 mM MgCl₂, 1 mM DTT, and 1 mg/ml L-cysteine. Proteins were transferred to nitrocellulose and immunoblotted with anti-His (MA1-21315 Thermo Fisher Scientific) 1:2000, and anti-CCT4 Rabbit Invitrogen 1:2500.

Proteins from fractions gathered from Ni–NTA chromatography were separated by SDS-PAGE (Laemmli 1970 (link)) followed by Western-blot analysis of 6xHis-tagged proteins. The gel was either dyed with Coomassie Brilliant Blue R250 or transferred onto a PVDF membrane using the Towbin et al. (1979 (link)) buffer system. Immunodetection was carried out with mouse anti-6xHis-tag monoclonal antibodies (ThermoFisher; MA1-21,315) as primary and goat anti-mouse antibody conjugated with alkaline phosphatase (Life Technologies; A16087) as secondary antibody. A solution with nitro blue tetrazolium chloride and 5-brom-4-chloro-3-indolyl-phosphate was used for visualization (https://www.sysy.com/protocols/westernblot-ap-detection). Proteins conjugated with a 6xHis-tag show up with a purple-colored band.

Culture samples were centrifuged at 200× g for 10 min and supernatants collected and stored at −80 °C until further analysis. Western blot analysis was performed as reported elsewhere [29 (link)]. For S identification, the human monoclonal antibody SARS-CoV-2 spike protein (MA5-35948, Thermo Scientific, Breda, The Netherlands) was used at dilution of 1:3000. For 6his-tag recognition on S proteins identification, a mouse monoclonal antibody anti-6His tag (MA1-21315, Thermo Scientific, Breda, The Netherlands) was used at a dilution of 1:1000. As secondary antibody, an anti-mouse IgG (A3438, Sigma, Amsterdam, The Netherlands) and an anti-human IgG antibody (A9544, Sigma, Amsterdam, The Netherlands) conjugated with alkaline phosphatase were used at a dilution of 1:5000.

Proteins were run on SDS–PAGE and were electrophoretically transferred onto a PVDF membrane. The transferred proteins on the PVDF were incubated with blocking solution containing 5% (w/v) non‐fat dried skimmed milk powder and TBST [0.1% Tween‐20 in Tris‐buffered saline; 137 mM NaCl and 20 mM Tris–HCl, (pH 7.4)] for 1 h at room temperature. Thereafter, the membranes were treated with anti‐MCU (HPA016480, Sigma), anti‐MCU (raised against a synthetic peptide (₃₂₈NEMDLKRLRDPLQVHLPLRQIGEKDC₃₅₁) from human MCU and named anti‐MCU‐C), anti‐MICU1 (HPA037480, Sigma), anti‐Flag (F‐3165 and F7425, Sigma), anti‐MICU2 (ab101465, Abcam), anti‐His (MA1‐21315, Thermo Scientific), anti‐GFP (sc‐9996, Santa Cruz), anti‐flavoprotein subunit of complex II (459200, Mito Sciences), anti‐myc (sc‐40, Santa Cruz) and anti‐GAPDH (LF‐MA0026, Lab Frontier) antibodies, washed three times with TBST and then incubated with horseradish peroxidase‐conjugated secondary antibody (111‐035‐006 and 115‐035‐006, Jackson). After washing, the membranes were treated with enhanced chemiluminescence solution (Pierce), and the signals were detected using an ImageQuant LAS 4000 (GE Healthcare Life Sciences).