Smurf1

Immunoprecipitation was performed as previously described.^{48 (link)} MC3T3-E1 cells or primary osteoblasts were treated with 5 μmol·L⁻¹ MG132 (Sigma) for 6 h. Cells were lysed in buffer (150 mmol·L⁻¹ NaCl, 50 mmol·L⁻¹ Tris, pH 7.8, 10% glycerol, 1 mmol·L⁻¹ EGTA, 0.5% NP-40, 1 mmol·L⁻¹ EDTA, 1 mmol·L⁻¹ PMSF, 1× cocktail) on ice with vigorous shaking for 30 min. The lysates were centrifuged at 4 °C at 12 000 r·min⁻¹ for 15 min. Twenty microliters of lysate was saved as the total protein sample. The rest of the supernatant was incubated with 20 μL of protein A/G (Santa Cruz) for 40 min to exclude nonspecific binding. After 10 min of centrifugation at 12 000 r·min⁻¹, the supernatant was subjected to IP with Runx2 antibody (Cell Signaling Technology, 12556S) and protein A/G at 4 °C for 4 h. The antibody and protein A/G were incubated for 2 h in advance. Finally, the protein A/G beads were collected and washed with cell lysis buffer twice. The lysates were subjected to western blotting with the indicated antibody. The following antibodies were used in the study: ubiquitin (1:2 000, Cell Signaling Technology, 3936S), GAPDH (1:5 000, Abways Technology, AB0036), Runx2 antibody (1:1 000, CST, 12556S), NONO (1:1 000, Abways Technology, CY8525), SFPQ (1:1 000, Abways Technology, CY8089), Smurf1 (1:1 000, Santa Cruz, sc-100616), and HA-tag (1:2 000, Cell Signaling Technology, #3724S).

Tissue sections and HAT‐7 (Osaka Dental University) cells were fixed, washed, blocked and incubated with antibodies as follows: Smurf1 (1:50, Santa Cruz, USA), F‐actin (Phalloidin‐iFluor 488, 1:1000, Abcam, USA), F‐actin (Phalloidin‐iFluor 594, 1:1000, Abcam, USA), RhoA (1:50, Proteintech, China), AMBN (1:50, Santa Cruz, USA), AMGN (1:50, Santa Cruz, USA), Alexa Fluor 594 goat anti rabbit (1:1000, Invitrogen, USA), Alexa Fluor 594 goat anti mouse (1:1000, Invitrogen, USA) and Alexa Fluor 488 goat anti mouse (1:1000, Invitrogen, USA). The images were observed and taken under fluorescence microscope (Olympus, Japan).

Immunoprecipitation and immunoblotting were carried out as described previously [46 (link)]. The following antibodies were used: IRF3 (1:1000, Santa Cruz, sc-9082), p-IRF3 (1:1000, Cell Signaling, 4947S), OTUD1 (1:1000, Abcam, ab182511), Flag (1:5000, Sigma, F7425), β-actin (1:5000, Proteintech, 66009-1-Ig), Myc (1:5000, Abmart, m2002), MAVS (1:1000, Santa Cruz, sc-166583), TRAF3 (1:1000, Cell Signaling, 4729S), TRAF6 (1:1000, Abcam, ab94720), TBK1 (1:1000, Cell Signaling, 3013S), RIG-I (1:1000, Cell Signaling, 4200S), Smurf1 (1:1000, Santa Cruz, sc-100616), Smurf2 (1:1000, Santa Cruz, sc-25511), Ub (1:1000, Santa Cruz, sc-8017), HA (1:5000, Abcam, ab9110), K48-Ub (1:1000, Cell Signaling, 4289S), and VSVG (1:5,000, Santa Cruz, sc-66180).