Anti ubiquitin sc 271289

Immunoblot analysis was performed as previously described^{29 (link)}. Cells were lysed with RIPA buffer (Beyotime). Protein samples were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Primary antibodies used include 1/1000 anti-Actin (AF5001), 1/1000 anti-GST (AF2299), 1/1000 anti-Flag (AF0036) (Beyotime), 1/1000 anti-HA (SAB2702196), 1/1000 anti-Myc (SAB2702192), 1/500 anti-SOCS7 (SAB2103430), 1/500 anti-IKKγ (SAB3500414) (Sigma-Aldrich), 1/500 anti-iNOS (2982), 1/200 anti-phospho-IκBα (Ser32/36) (9246), 1/500 anti-IκBα (4814), 1/200 anti-phospho-IRF-3 (Ser386) (37829), 1/500 anti-IRF-3 (11904), 1/200 anti-phospho-STAT2 (Tyr690) (4441), 1/500 anti-STAT2 (4594), 1/500 anti-NF-κB p65 (8242), 1/500 anti-IKKα (2682), 1/500 anti-IKKβ (2678), 1/500 anti-phospho-IKKα/β (Ser176/180) (2697) (Cell Signaling Technology, Inc., Danvers, MA, USA), 1/500 anti-Ubiquitin (sc-271289), 1/200 anti-PTPN14 (Pez) (sc-373766) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1/500 anti-STAT1 (AHO0832), and 1/200 anti-phospho STAT1 (Tyr701) (44–376G) (Invitrogen). Immunoblots were revealed using the SuperSignal west pico substrate (ThermoFisher Scientific, San Jose, CA, USA). For coimmunoprecipitation (Co-IP), the lysates were incubated with appropriate antibodies and Protein A/G beads (ThermoFisher Scientific) overnight at 4 °C followed by immunoblot analysis.

MDA-MB-231, MDA-MB-468, and MCF-10A cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and the mouse embryonic fibroblasts (MEFs) were received from Dr. Steven Frisch (West Virginia University). With regard to human mammary luminal epithelial (HMLE) cells, only cell lysates received from Dr. Alexey Ivanov (West Virginia University) were used. The conditions for cell growth were described previously [21 (link), 25 (link)]. Antibodies used in the study were anti-EGFR (610017) and anti-SHP2 (610822) from BD Biosciences (San Jose, CA, USA); anti-CBL (sc-1651), anti-pan-extracellular signal-regulated kinase 2 (anti-pan-ERK2; sc81457), and anti-ubiquitin (sc271289) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-β-actin (A5441) from Sigma-Aldrich (St. Louis, MO, USA); and anti-phospho-ERK1/2 (9101S), anti-phospho-Akt (9271S), and anti-pan-Akt (11E7) from Cell Signaling Technology (Danvers, MA, USA).