Table 1.
Names, sources, dilutions, and validation of antibodies used
| Antibody Name (abbreviation) | Source (catalog no.) | Dilution | Validation |
|---|---|---|---|
| Anti-Akt | Cell Signaling Technology (#4691) | 1:1,000 | • Validated using cells transfected with siRNA (38) • Widely cited (∼2,600 citations) |
| Anti-AS160 | MilliporeSigma (#ABS54) | 1:1,000 | Validated using muscles from wild-type and AS160-KO rats (39, 40) |
| Anti-γ3-AMPK | Dr. David Thomson, Brigham Young Univ., Provo, UT | 1:1,000 | Validated using muscles from wild-type and γ3-AMPK-KO mice (24) |
| Anti-GLUT4 | MilliporeSigma (#CBL243) | 1:100 | Validated using muscles from rats injected with AAV-GLUT4 vs. muscles from rats with sham injection (unpublished data from G.D.C.) |
| Anti-phospho Akt Ser473 (pAktSer473) | Cell Signaling Technology (#4060) | 1:1,000 | • Vendor validated antibody using cells stimulated by PDGF ± specific inhibitors (LY294002/wortmannin) • Widely cited (∼5,600 citations) |
| Anti-phospho Akt Thr308 (pAktThr308) | Cell Signaling Technology (#13038) | 1:1,000 | • Vendor verified antibody using cells stimulated with PDGF • Widely cited (∼500 citations) |
| Anti-phospho AMPKα Thr172 (pAMPKThr172) | Cell Signaling Technology (#2535) | 1:1,000 | Vendor validated antibody using cells treated with oligomycin |
| Anti-phospho AS160 Ser588 (pAS160Ser588) | Cell Signaling Technology (#8730) | 1:1,000 | • Vendor validated antibody phosphospecificity by λ phosphatase treatment. • Validated using AS160-KO rat muscles injected with AAV-AS160 with Ser588 mutated to Ala588 vs. muscles from wild-type rats with sham injection (unpublished data from G.D.C.) |
| Anti-phospho AS160 Thr642 (pAS160Thr642) | Cell Signaling Technology (#4288) | 1:1,000 | • Vendor validated antibody phosphospecificity by λ phosphatase treatment • Validated using AS160-KO rat muscles injected with AAV-AS160 with Thr642 mutated to Ala642 vs. muscles from wild-type rats with sham injection (unpublished data from G.D.C.) |
| Anti-phospho AS160 Ser704 (pAS160Ser7104) | J.T.T. | 1:1,250 | • Validated using mouse muscles with gene electrotransfer of flag-tagged AS160 with Ser704 mutated to Ala704 vs. mouse muscles with electrotransferred flag-tagged wild-type AS160 (unpublished data from J.T.T.) • Validated using AS160-KO rat muscles injected with AAV-AS160 with Ser704 mutated to Ala704 vs. muscles from wild-type rats with sham injection (unpublished data from G.D.C.) |
| Anti-rabbit IgG, HRP-linked | Cell Signaling Technology (#7074) | 1:20,000 | Widely cited (∼7,600 citations) |
AAV, adeno-associated virus; AMPK, AMP-activated protein kinase; AS160, Akt substrate of 160 kDa; GLUT4, glucose transporter type 4; HRP, horseradish peroxidase; KO, knockout; PDGF, platelet-derived growth factor.