Journal: Neurobiology of aging
Article Title: Insulin and adipokine signaling and their cross-regulation in postmortem human brain
doi: 10.1016/j.neurobiolaging.2019.08.012
Figure Lengend Snippet: Representative Western blots verifying completeness and specificity of immunoprecipitation for the frontal cortical proteins studied in postmortem human brains: IRβ (A), AKT1 (B), APPL1 (C), AMPKα1/2 (D), JAK2 (E) and STAT3 (F). Three postmortem human frontal cortices from control subjects were chosen for testing. Results shown are the representative of three samples. The lanes in each blot show relative amount of antigen in the tissue lysate, the immunoprecipitate (IP) of that lysate, and the remaining supernatant. The solubilized lysate was used to assess the specificity of immunoprecipitation using indicated antibody, IgG and protein A/G beads. Further, the resultant supernatant following IP was IP with specific antibodies. Results show at least 90% of each antigen was immunoprecipitated and each antibody was specific to the antigen indicated. N = 3
Article Snippet: Ratios of phosphorylated to total levels of each antigen were calculated. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Antibody (Ab) Ab Type Ab dilution AdipoR1 Santa Cruz 518,030 Mouse mAb 1:1000 AdipoR2 Santa Cruz 514045 Mouse mAb 1:1000 AKT1–3 Santa Cruz 8312 Rabbit pAb 1:500 pS 473 AKT1–3 Santa Cruz 514032 Mouse mAb 1:750 pT 308 AKT1–3 Santa Cruz 271964 Mouse mAb 1:750 AMPKα1/2 Santa Cruz 74461 Mouse mAb 1:750 pT 183/172 AMPKα1/2 Abcam 23875 Rabbit pAb 1:1000 APPL1 Santa Cruz 271,909 Mouse mAb 1:750 IRβ Santa Cruz 81465 Mouse mAb 1:500 pY 960 IRβ Invitrogen 44–800G Rabbit pAb 1:1000 pY 1150/1151 IRβ Santa Cruz 81500 Mouse mAb 1:1000 IRS-1 Santa Cruz 8038 Mouse mAb 1:750 JAK2 Santa Cruz 390,539 Mouse mAb 1:750 pY 1007/1008 JAK2 Cell Signaling #3776 Rabbit mAb 1:1000 OB-R Santa Cruz 8391 Mouse mAb 1:750 STAT3 Santa Cruz 8019 Mouse mAb 1:750 pY 705 STAT3 Santa Cruz 8059 Mouse mAb 1:750 T-Cadherin Santa Cruz 166875 Mouse mAb 1:750 Open in a separate window Key: AdipoR1, adiponectin receptor 1; AdipoR2, adiponectin receptor 2; AMPK, adenosine monophosphate-dependent protein kinase; APPL1, adaptor protein containing pleckstrin homology domain phosphotyrosine-binding domain and leucine zipper motif; OB-R, leptin receptor; JAK2, Janus kinase 2; IR, insulin receptor; STAT3, signal transducer and activator of transcription 3; pY, phosphorylated; IRS-1, insulin receptor substrate-1.
Techniques: Western Blot, Immunoprecipitation
