phospho tyr612 insulin receptor substrate 1 irs1  (Cell Signaling Technology Inc)

Journal: Diabetes & Metabolism Journal

Article Title: Extracellular Vimentin Alters Energy Metabolism And Induces Adipocyte Hypertrophy

doi: 10.4093/dmj.2022.0332

Figure Lengend Snippet: Extracellular vimentin-induced glucose transporter type 1 (GLUT1) expression depends on insulin-like growth factor 1 receptor (IGF1R) and activation of extracellular-signal-regulated kinase (ERK). (A) Western blot analyses for phospho-insulin receptor substrate 1 (P-IRS1; Tyrosine 612), P-Akt (Serine 473), and P-IGF1R (Tyrosine 1158+1162+1163) were performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Samples from three different batches of treatment were loaded onto a gel ( n =3). (B) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for GLUT1, GLUT4, hypoxia-inducible factor 1α (Hif-1α) were performed with RNA from 3T3-L1-derived adipocytes treated or untreated with anti-IGF1R antibody (11 µg/mL) for 1 hour and then incubated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (GLUT1, n =3; GLUT4, n =3; Hif-1α, n =4). (C) Western blotting for P-ERK1/2 (Threonine 202/Tyrosine 204) was performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using GAPDH as an internal control ( n =3). (D) qRT-PCR analyses for GLUT1, GLUT4, Hif-1α were performed with RNA from 3T3-L1-derived adipocytes treated or untreated with U0126 (ERK inhibitor, 25 µM) for 1 hour and then incubated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control ( n =3 for each). a P <0.05, b P <0.01, c P <0.001.

Article Snippet: Antibodies for GLUT1, GLUT4, phospho-insulin receptor substrate 1 (P-IRS1), P-Akt, phospho-extracellular-signal-regulated kinase (P-ERK), phospho-hormone-sensitive lipase (P-HSL; S563), HSL, peroxisome proliferator-activated receptor γ (PPARγ), P-ERK, inositol-requiring enzyme 1 α (IRE1α), C/EBP homologous protein (CHOP), p62, and microtubule-associated proteins 1A/1B light chain 3 (LC3)-I/II were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Activation Assay, Western Blot, Derivative Assay, Recombinant, Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Incubation