Anti k63 linkage specific polyubiquitin d7a11

The following antibodies were used for Western blots: anti-murine Mpeg1 (ab25146), anti-human Mpeg1 (ab176974), anti-Cullin-1 (Ab75817), anti-GFP (ab290), and anti-β-TRCP (Ab137674) (Abcam, Cambridge, MA); anti-GFP (sc9996) (Santa Cruz Biotechnology, Dallas, TX); anti-β-actin (2F1-1); anti-Ubiquitin (P4D1) (BioLegend, San Diego, CA, United States); anti-Ubc12 (D13D7); anti-K63-linkage specific polyubiquitin (D7A11); anti-K48-linkage specific polyubiquitin (D9D5) (Cell Signaling Technology, Danvers, MA, United States) and anti-FLAG (F-7425) (Sigma-Aldrich). Densitometry analysis was performed where indicated utilizing ImageJ software. Co-IP and ubiquitylation assays were modified from (Shembade et al., 2010 (link)) utilizing Dynabeads Co-immunoprecipitation kit (Thermo Fisher Scientific, Waltham, MA, United States). Whole-cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked in 5% milk, incubated with specific primary and secondary antibodies, then detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) by either X-ray film or Odyssey FC Imaging System (LI-COR, Lincoln, NE, United States).

Anti-IRAK1 (D51G7) (Cat#4504), Anti-TRAF6 (D21G3) (Cat#8028), anti-Myc (9B11) (Cat#2276), anti-ubiquitin (P4D1) (Cat#3936), anti-K48-linkage-specific polyubiquitin (D9D5) (Cat#8081), anti-K63-linkage-specific polyubiquitin (D7A11) (Cat#5621), anti-p65 (Cat#8242), and anti-phospho-p65 (Ser536) (Cat#3033) were purchased from Cell Signaling Technology (Beverly, MA, USA).Anti-TRAF6 (Cat#ab137452) was purchased from Abcam (Cambridge, MA, USA). Anti-Flag (Cat# F3165) and anti-HA (Cat# H6908) antibodies were purchased from Sigma-Aldrich. Anti-TRAF6 (Cat# 66498-1-lg) and anti-GAPDH (Cat# 60004-1-lg) were purchased from Proteintech (Wuhan, Hubei, China). Anti-Rabbit IgG FITC (Cat# A22120) and anti-Mouse IgG DyLight 649 (Cat# A23610) antibodies were purchased from Abbkine.