Anti phospho her2 erbb2 tyr1248

A 20 μg protein aliquot was resuspended in Laemmli buffer, resolved on polyacrylamide gels under reducing conditions and transferred to polyvinylidine difluoride membranes (Immobilon-P, EMD Millipore Corporation). Membranes were blocked with 5% skim milk or 5% BSA in Tris buffer saline (TBS) with 0.1% Tween-20 (Sigma-Aldrich) for 1 h at room temperature, and incubated with appropriate primary antibodies: anti-phospho-HER2/ErbB2 Tyr1248 (Cell Signaling Technology, Inc.), anti-HER2/ErbB2 (clone 29D8, Cell Signaling Technology, Inc.), anti-p27 KIP1 (Abcam), or anti-α-tubulin (Sigma-Aldrich). Antibodies conjugated to horseradish peroxidase (Abcam) were used as secondary antibodies, and chemiluminescence reaction was developed with the ECL star kit (Euroclone). Densitometric analysis of protein bands was performed with ImageJ software.

The following drugs were purchased: Dinaciclib (SCH727965) for in vitro and in vivo studies (S2768; Selleckchem), lapatinib ditosylate (Tykerb) for in vitro and in vivo studies (M1802; Abmole), neratinib for in vivo studies (M1913; Abmole), A-1210477 (CT-A121; Chemietek), A-1331852 (22963; Cayman Chemicals), tucatinib (HY-16069; Medchem), and ABT-199 (venetoclax) (CT-A199; Chemietek). The antibodies used in this study were as follows: anti-Bak (AB-1 clone for IP) (AM03; EMD Millipore), anti-Bak (3814S; Cell Signaling), anti-Bim (C34C5) (2933S; Cell Signaling), anti–BCL-xL (54H6) (2764S; Cell Signaling), anti–Bcl-2 (D55G8) (Human Specific) (4223S; Cell Signaling), anti-cleaved PARP (Asp214) (D64E10) (5625S; Cell Signaling), anti-GAPDH (6C5) (sc-32233; Santa Cruz), anti–MCL-1 (S-19) (sc-819; Santa Cruz), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (4370S; Cell Signaling), anti-phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) (5364S; Cell Signaling), anti-phospho-Akt (Thr308) (244F9) (4056S; Cell Signaling), anti-phospho-Akt (Ser473) (D9E) (4060S; Cell Signaling), anti-HER2/ErbB2 (29D8) (2165S; Cell Signaling), anti-phospho-HER2/ErbB2 (Tyr1248) (2247S; Cell Signaling), anti-phospho-Rpb1 CTD (Ser 2/5) (4375S; Cell Signaling), Normal Rabbit IgG for IP (sc-2027; Santa Cruz), and Normal Mouse IgG for IP (sc-2025; Santa Cruz).