Anti phospho akt ser473 9271

Western blot analyses were performed as previously described [42 (link)]. Antibodies used were anti-Fra-1 (R-20; sc-605), anti-c-Jun (H-79; sc-1694), anti-p-c-Jun (KM-1; sc-822) and anti-ZEB2 (E-11; sc-271984) from Santa Cruz Biotechnology; anti-E-cadherin (610182), anti-N-cadherin (610920), and anti-fibronectin (610077) from BD Biosciences; anti-β-actin from Sigma; anti-p-Fra-1 (Ser265; #3880), anti-Akt #9272, anti-phospho-Akt (Ser473; #9271), anti-ERK, and anti-phospho-ERK were from Cell Signaling Technology.

Protein extracts from cells or tissues were prepared by homogenization in RIPA lysis buffer (Santa Cruz). Protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membrane (Millipore). After incubation with the indicated primary antibodies at 4 °C overnight, the blots were incubated with IR dye-coupled secondary antibodies (LI-COR) and visualized by the LI-COR Odyssey infrared imaging system. The anti-FLAG (F3165) and anti-β-actin (A1978) antibodies were from Sigma. The anti-phospho-Akt (Ser473) (9271) and anti-Akt (2920) antibodies were from Cell Signaling Technology. Full-length western blot images corresponding to the data presented in main/supplementary figures are provided in Supplementary Figure 13.

Metformin was obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-phospho (Ser473) Akt (#9271) and total Akt (#9272) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against GLUT4 (sc-7938), p-IRS1 (Tyr632) (sc-17196), IRS1 (sc-559), p-PI3K (Tyr508) (sc-12929), PI3K (sc-7174), PGC1α (sc-13067), PPARα (sc-9000), UCP1 (sc-6529), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-365062) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). FGF21 (ab64857) was purchased from Abcam (Cambridge, UK).

We used the following primary antibodies as follows: anti-Akt (9272, Cell Signaling) at 1:1000; anti-phospho(Ser473)-Akt (9271, Cell Signaling) at 1:1000; anti-cFos (PC38, Calbiochem) at 1:10000; anti-Darpp32 (AB10518, Millipore) at 1:2500; anti-ERα (06–935, Millipore) at 1:500; anti-Parvalbumin (PVG214, Swant) at 1:5000. In the immunostaining procedure, we used secondary antibodies anti-rabbit IgG and anti-goat IgG conjugated with Alexa Fluor 488 or Alexa Fluor 568 at 1:1000 (Invitrogen), AffiniPure Fab Fragment Goat Anti-rabbit IgG (H+L) (111-007-003, Jackson Laboratories). In the Western blot protocol, we used HRP-conjugated secondary antibodies anti-rabbit IgG (31460, Thermo Scientific) and anti-mouse IgG (NA931V, GE Healthcare) at 1:10000. The specificity of each antibody used in this study has been extensively tested by other groups and showed specific and expected staining in our hands.
For quantitative PCR, we used the following TaqMan probes (from Thermofisher): Gdnf1-2 (Gdnf exons 1 to 2), Mm00599849_m1; Pvalb (parvalbumin), Mm00443100_m1; Actb (βActin), Mm00443100_m1; Gapdh, Mm99999915_g1; Hmbs, Mm00660262_g1.
17β-estradiol (E8875, Sigma) was used to make up the SILASTIC implants, and β-estradiol 3-benzoate (E-8515, Sigma) for subcutaneous injection.