Anti pan ras

In vivo ubiquitylation assays and immunoblot analyses were performed as previously described [19 (link)]. Briefly, N-ethylmaleimide (10 mM, Sigma) was added to the radioimmunoprecipitation assay (RIPA) buffer (Upstate Biotechnology). The lysates were incubated with the indicated antibodies and Protein G agarose at 4°C for 12 h, and the beads were washed three times with cold RIPA buffer. Immunoblot analysis was performed as previously described [19 (link)]. Primary antibodies were obtained from the following sources; anti-pan-Ras (Millipore); -K-Ras, -N-Ras, -H-Ras, -β-actin, -p-ERK, -p-AKT, -PKCδ, -PKCα,-PCNA, and -c-Myc (Santa Cruz Biotechnology); -HA, -PKCε, and -PKCζ (Cell Signaling Technology); -Flag (Sigma); and -ER-α36 (Cell Applications). Secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse (Cell Signaling Technology) and HRP-conjugated anti-rabbit (Bio-Rad).

The antibodies used in this study were anti-p-ATF2 (T71) (Cell Signaling Technology); p-EGFR (Y1068) (Cell Signaling Technology); p-ERK (T202/Y204) (Cell Signaling Technology); α-tubulin (Calbiochem); β-catenin (BD Biosciences and Santa Cruz Biotechnology); caspase-3 (Santa Cruz Biotechnology); EGFR (Cell Signaling Technology); ERK1 (Santa Cruz Biotechnology); PARP-1/2 (Santa Cruz Biotechnology); PCNA (Santa Cruz Biotechnology); anti-Ki67 (Abcam); anti-Pan-Ras (Millipore); anti-rabbit AlexaFluor 488 (Life Technologies). Erlotinib (Tarceva®) was purchased from Cayman. KYA1797K was described in our previous study^{14 (link)}. Cycloheximide was purchased from Sigma-Aldrich.

Cells were washed once with PBS and lysed in SDS-Tris buffer (1% SDS, 10 mM Tris-HCl pH 7.4) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Thermo-Fisher). Cell lysates were sonicated with a Branson Sonifier and the protein concentrations determined by using the Pierce BCA protein assay kit (Thermo-Fisher). Equal amounts of protein (10 μg) were resolved on 10 or 15% SDS-PAGE and subsequently transferred onto a PVDF membrane (GE). The membrane was blocked either with 10% non-fat milk (Sigma) or 10% BSA (Sigma) in TBS-0.1% Tween20 and incubated overnight with primary antibody at 4°C. After washing the membrane was incubated with HRP conjugated secondary antibody for 1 hr at room temperature (RT, 25°C). The membrane was washed with TBS-0.1% Tween and developed using Pierce ECL Western Blotting Substrate (Thermo-Fisher) and CL-XPosure films (Thermo-Fisher). Primary antibodies include anti-phospho-p44/22 MAPK (ERK1/2) (CST), anti-p44/42 MAPK (total ERK1/2) (CST), anti-phospho-MEK1/2 (CST), anti-MEK1/2 (CST), anti-phospho-AKT S473 (CST), anti-AKT (CST), anti-pan-RAS (Millipore), anti-GFP (Santa Cruz Biotechnologies), anti-β-actin (Sigma). Secondary antibodies include anti-CMYC HRP-linked (Novus Biologicals), anti-mouse IgG HRP-linked (CST) and anti-rabbit IgG HRP-linked (CST).