Anti irs1

For Western blot analysis, the following antibodies were used: anti-Pol II CTD (2629S, Cell Signaling Technology, Danvers, MA, USA), anti-Pol II p-CTD (Ser2) (13499S, Cell Signaling Technology), anti-cyclinK (A301-939A-M, Bethyl Laboratories), anti-IRS1 (3407S, Cell Signaling Technology), anti-WNT1 (SC-514531, Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin (MAB1501R, Millipore, Burlington, MA, USA), and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse GTX213112-01; anti-rabbit GTX213110-01) (Genetex, Irvine, CA, USA).

As described previously [4 (link)], gastrocnemius muscle and the liver samples were processed by homogenizing in ice-cold RIPA buffer (Beyotime P0013C, China) supplemented with complete protease inhibitor cocktail (Roche, Germany) and PhosSTOP (Roche, Germany). The protein concentration of the supernatant obtained by centrifugation was determined with a BCA protein assay kit (Pierce Biotechnology, Rockford). The protein extracts (40 μg) for each preparation were separated by SDS-PAGE and analyzed by specific antibodies including anti-AKT (Cell Signaling Technology, Cat no. #4685, Beverly, MA, USA), anti-phospho-AKT Ser⁴⁷³ (Cell Signaling Technology, Cat no. #4058, Beverly, MA, USA), anti-IRS1 (Cell Signaling Technology, Cat no. #2382, Beverly, MA, USA), and anti-phospho-IRS1 (Ser³⁰⁷) (Cell Signaling Technology, Cat no. #2381, Beverly, MA, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies, the immunoblots were visualized with an ECL Kit (WBKLS0050; Millipore, Billerica, MA, USA), and quantitated by densitometric analysis using ImageJ.

RIPA buffer (Sigma, St. Louis, MO, USA) containing Protease Inhibitor Cocktail (GenDEPOT) was used for cell lysis. Cell lysates were boiled with lane markers in reducing sample buffer (Thermo Fisher Scientific, Waltham, MA, USA). The proteins in cell lysates were separated via SDS/polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. The membrane was then blocked for 1 h with 5% skim milk/Tris-buffered saline and Tween 20 (TBST). Next, the membrane was incubated overnight at 4 °C with a primary antibody (diluted 1:1000 in 5% skim milk/TBST). The membrane was washed three times with TBST buffer and then incubated for 1 h at room temperature with an HRP-conjugated secondary antibody (1:5000 in 5% skim milk/TBST). Finally, the membrane was washed three times with TBST buffer and incubated with West-Q Pico ECL solution and West-Q Femto clean ECL solution (GeneDEPOT, Baker, TX, USA). Anti-IRS1 (Cell Signaling Technology, Trask Lane Danvers, MA, USA) and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) were used as primary antibodies. HRP-conjugated antirabbit IgG or HRP-conjugated anti mouse IgG were used as secondary antibodies.

Immunocytochemistry was performed as previously described [26 (link)]. Briefly, after fixing cells with 4% paraformaldehyde in phosphate buffered solution (PBS) (Cat. # AAJ19943K2, Thermo Fisher Scientific Inc.), they were permeabilized by adding cold methanol and then probed with the indicated primary antibodies. Antibodies used are as follows: anti-α-synuclein (#2642, 1:1000, Cell Signaling Technology), anti-phospho-α-synuclein (Ser129) (ab51253, 1:1000, Abcam plc.), anti-tyrosine hydroxylase (MAB318-AF488, 1:100, Millipore), anti-FOXA2 (ab108422, 1:300, Abcam plc), anti-phospho-IRS1 (Ser302) (Cat. # 2384, 1:1000, Cell Signaling Technology), and anti-IRS-1 (Cat. # 2382, 1:1000, Cell Signaling Technology). Fluorescence intensities from images of randomly selected microscopic fields of cells were semi-quantitatively analyzed based on staining positivity indices estimation using the NIH ImageJ software (https://imagej.nih.gov/ij/) as previously described by Jensen [27 (link)].

The following commercially available primary antibodies were used for immunoblotting: anti-IRS2 (Cell Signaling Technologies, 4502) 1:750; anti-Cdh1/Fzr1 (Sigma Aldrich, CC43) 1:500; anti-anillin (a gift from Christine Field (21 (link))) 1:1000; anti-Aurora B (Bethyl, A300-431) 1:1000; anti-Eg5 (Cell Signaling Technologies, 4203) 1:1000; anti-Top2A (Cell Signaling Technologies, 12286) 1:1000; anti-TK1 (Cell Signaling Technologies, 8960) 1:1000; anti-Mps1 (Abcam, ab11108), 1:1000); anti-APC3 (BD Transduction Laboratories, 610455) 1:500; anti-cyclin B1 (Santa Cruz Biotechnology, sc-752) 1:500; anti-Cdc20 (Santa Cruz Biotechnology, sc-8358) 1:500; anti-c-Myc (9E10, Santa Cruz Biotechnology, sc-40) 1:1000; anti-HA-peroxidase (Sigma Aldrich), 1:1500; anti-cyclin A2 (Santa Cruz Biotechnology, sc-596) 1:500; anti-IRS1 (Cell Signaling Technologies, 2382) 1:750; anti-MyoD1 (Cell Signaling Technologies, 13812) 1:750; anti-GAPDH (Abcam, ab8245) 1:2000; anti-α tubulin (Abcam, ab7291 and Santa Cruz Biotechnology, sc-8035) 1:1000 for both; anti-vinculin (Santa Cruz Biotechnology, sc-73614) 1:2000. Secondary antibodies used: anti-rabbit IgG-HRP (GE Healthcare, NA934) and anti-mouse IgG-HRP (GE Healthcare, NA931V), both at 1:3000 dilutions.

The antibodies used were anti-Akt (rabbit polyclonal, #9272), anti-phospho-Akt (rabbit polyclonal, #9271), anti-β-Actin (rabbit polyclonal, #4967), anti-IRS1 (rabbit polyclonal, #2382) and anti-phospho-IRS1^ser307 (rabbit polyclonal, #2381) from Cell Signaling Technology (Beverly, MA, USA); anti-JNK (rabbit polyclonal, SC1648), anti-phopho-JNK (mouse monoclonal, SC6254), anti-G6pase (goat polyclonal, SC 27198) and anti-α-Tubulin (mouse monoclonal, SC8035) from Santa Cruz Technology (Santa Cruz, CA, USA). Atorvastatin was obtained from Pfizer (Loughbeg, County Cork, Ireland) and Diacerein was kindly provided by TRB-Pharma (Campinas, Brazil). Human recombinant insulin was from Eli Lilly and Co. (Indianapolis, Indiana, USA). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless specified elsewhere.

Anti-NDRG1 antibody was generated as previously described [43 (link)]. Other antibodies were purchased as follows: anti-α-tubulin antibody was from Sigma-aldrich Co; anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-phospho-HER3, anti-Met, anti-phospho-Met, anti-PDGFRβ, anti-phospho-PDGFRβ, anti- IGF-1Rβ, anti-phospho-IGF-1Rβ, anti-ERK1/2, anti-phospho-ERK1/2, anti-AKT, anti-phospho-AKT (Thr308 and Ser473), anti-mTOR, anti-phospho-mTOR (Ser2448 and Ser2481), anti-Raptor, anti-Rictor, anti-S6K, anti-phospho-S6K(Thr389), anti-phospho-S6, anti-PTEN, anti-phospho-PTEN, anti-IRS-1, anti-PDK1, anti-PARP, anti-phospho-PDK1, anti-TSC-1, anti-TSC-2, and anti-phospho-4EBP-1 antibodies were from Cell Signaling Technology (Beverly, MA); anti-HER2 and anti-HER3 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Trevigen Inc. (Gaithersburg, MD): anti-p27 antibody was from BD Biosciences (San Jose, CA): anti-cleaved PARP antibody was from Promega (Madison, WI): anti-phospho-HER2 antibody was from Millipore (Billerica, MA): anti-β-actin was from Abcam (Cambridge, UK).