Sc 197

Specified antibodies to the following proteins were used for immunoblotting (IB), immunoprecipitation (IP), immunofluorescence (IF), and immunohistochemistry (IHC): phospho-Akt1(S473) (1:2000, IB; ab81283, abcam), phospho-Bad(S112) (1:1000, IB, no. 5284, Cell Signaling), phospho-BIM(S69) (1:400, IB; no. 4581, Cell Signaling), phospho-Elk1 (S383) (1:1000, IB; no. 9181, Cell Signaling), phospho-Elk1 (S383) (1:1000, IB; ab34270, abcam), phospho-ERK1/2(TEY) (1:1000, IB; no. 9101L, Cell Signaling), ERK1/2 (1:1000, IB; no. 9102, Cell Signaling), phospho-ERK1/2(T188) (1:1000, IB; 1:500 IHC; Lorenz et al., 2009 (ref. ^{13 (link)}) or A010-40AP, Badrilla), Flag (1:10,000, IB or 1:200, IF; IP 5.6μg/sample;; F3165, Sigma), phospho-Foxo3a(S294) (1:1000, IB; no. 5538, Cell Signaling), Gβ (1:5000, IB; sc-378, Santa Cruz Biotechnology), HA (1:5000, IB; IP 5μg/sample; MMS-101R, Covance), HA (1:500, IF; no. 3724, Cell Signaling), Max (1:200, IF; sc-197, Santa Cruz Biotechnology), c-Myc (1:1000, IB; sc-789, Santa Cruz Biotechnology), c-Myc (1:350, IF; M4439, Sigma), phospho-p70(S6) (1:1000, IB; no. 9204, Cell Signaling), and phospho-p90RSK(S380) (1:5000, IB; ab32203, abcam).

Total protein was isolated as previously described.^{23 (link)} Twenty microgram of total protein for MAX and 10 μg for MYC were separated by electrophoresis on 10% Mini-Protean gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes using a Semi-dry Trans-Blot System (Bio-Rad). The proteins were blocked (Tris-buffered saline with Tween 20 and 5% bovine serum albumin for 4 h at room temperature (RT)) and incubated overnight at 4°C with a MAX antibody (1:2000; sc-197, Santa Cruz Biotechnology, Santa Cruz, CA, USA)^{24 (link)} or a MYC antibody (1:1000; sc-40).^{25 (link)} The blots were then incubated with secondary antibodies (1:4000; sc-2004 or sc-2005, 1 h at RT). For the loading control, the membranes were stripped with 5% acetic acid for 5 min at RT, blocked with 5% bovine serum albumin for 1 h and incubated with anti-β-Actin (1:1000; sc-47778) or anti-γ-tubulin (1:10 000; T6557, Sigma-Aldrich, St. Lewis, WA, USA) antibodies overnight at 4 °C. The protein levels were detected with enhanced chemiluminescence (Bio-Rad) and analyzed with ImageQuant 7.0 (GE Healthcare Life Sciences, Piscataway, NJ, USA). The levels of both loading control proteins (β-actin and γ-tubulin) were similar between groups. The optical densities of the MAX and MYC bands were determined by normalization to the corresponding control bands.