Imagequant las 4000 chemiluminescent image analyzer

HEK293T cells that were transfected for 40 h with FLAG-MDA5 (G74A/W75A) together with WT or mutant ISGylation machinery components were lysed in Nonidet P-40 (NP-40) buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% (v:v) IGEPAL® CA-630, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 × protease inhibitor cocktail (MilliporeSigma)). The cell lysates were cleared by centrifugation at 20,000 × g and 4 °C for 20 min and then subjected to FLAG pulldown at 4 °C for 16 h using Dynabeads Protein G (Thermo Fisher Scientific) pre-conjugated with the anti-FLAG M2 antibody (MilliporeSigma). The beads were extensively washed with NP-40 buffer and proteins were eluted by heating in 1 × Laemmli SDS sample buffer at 95 °C for 5 min. Protein samples were resolved on Bis–Tris SDS-polyacrylamide gel electrophoresis (PAGE) gels, transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad), and visualized using the SuperSignal West Pico PLUS chemiluminescence reagents (Thermo Fisher Scientific) on an ImageQuant LAS 4000 Chemiluminescent Image Analyzer (General Electric) as previously described^{7 (link)}. The antibodies used for immunoblotting include: anti-V5 (1:1,000, SV5-Pk1; Invitrogen), anti-HA (1:1,000, C29F4; Cell Signaling Technology), anti-FLAG (1:1,000, M2; MilliporeSigma), anti-Myc (1:1,000, 9B11; Cell Signaling Technology), and anti-β-Actin (1:500, C4; Santa Cruz).

Tissues or cells were lysed on ice with precooled lysis buffer for 30 min and centrifuged at 12 000 rpm for 10 min at 4 ℃. The supernatant was transferred to centrifuge tubes, and 5 × SDS loading buffer was added. Samples were denatured for 10 min at 99 ℃ and subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Merck). The membrane was blocked with 5% BSA for 60 min at room temperature and incubated with the primary antibodies against Serpine1 (1:1000; Abcam), Nr4a1 (1:1000, Abcam), β-actin (1:1500, Yeasen), Gapdh (1:1500, Yeasen), and transferrin (1:1000, Beyotime) overnight at 4 ℃. After washing three times with 1 × TBST, the membrane was probed with the HRP-linked goat anti-rabbit secondary antibody (1:1000, Yeasen) for 60 min at room temperature and washed with 1 × TBST buffer. Proteins were visualized using electrogenerated chemiluminescence reagents (Epizyme) and detected with an ImageQuant LAS 4000 chemiluminescent Image Analyzer (General Electric). The relative densitometric density of the target band to the internal reference band was analysed using the ImageJ software.