Anti p tyr

M2 anti-FLAG antibody, cyclohexamide (CHX), and CKII inhibitor, DMAT, were purchased from Sigma-Aldrich (Oakville, ON, Canada). Recombinant human EGF and V5 antibody were from Life Technologies (Burlington, ON, Canada). Anti-p-Tyr, anti-GAPDH and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and used to detect non-specific phosphorylated tyrosine residues. Purified calpains, calpeptin, N-Ac-Leu-Leu-norleucinal (ALLN), and A23187 were purchased from Calbiochem-Novabiochem (San Diego, CA, USA). A kinase inhibitor library was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Calf intestinal phosphatase (CIP) was purchased from New England Biolabs (Whitby, ON, Canada). GF109203X and NU 112455A were from Selleckchem (Houston, TX) and EMD Millipore (Etobicoke, ON), respectively.

A. fumigatus extracts (AFE) was purchased from GREER (Lenoir, NC). AG1478 (Sigma, St. Louis, MO), BIBX 1382 (Sigma, St. Louis, MO), neutralizing anti-EGFR antibody (Calbiochem, La Jolla, CA), Raf-1 inhibitor (Sigma, St. Louis, MO) and sorafenib (LC laboratories, Woburn, MA), U0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene) (Sigma, St. Louis, MO). PMSF and Glutathione reduced ethyl ester (GSH-MEE) were from Sigma (St. Louis, MO) and phosphatase inhibitor was from Thermo Fisher Scientific (Waltham, MA). Antibodies targeting pERK1/2, pRaf-1, and anti-PAR2 neutralizing antibody were purchased from Cell Signaling Technology (Danvers, MA). Anti-MUC5B, anti-pEGFR (Y1173), anti-EGFR, anti-pTyr and β- ACTIN antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), Anti-MUC5AC and anti-Ras antibodies were obtained from Thermo Fisher Scientific (Grand Island, NY). Protease assay kit was obtained from Invitrogen (Carlsbad, CA).

Male mice (6–8 weeks old; n = 6–7/group) were randomised to a standard diet (16.7% kJ fat and 12.4% kJ sugar wt/wt) or high-fat/high-sugar diet (HFHS; 49.2% kJ fat and 32.2% kJ sugar wt/wt) for 15 weeks. Body weight and food consumption were measured weekly. At the end of the study, GTTs and metabolic measurements were performed. Metabolic measurements were measured using a Columbus Instruments Comprehensive Lab Monitoring System (CLAMS; Columbus Instruments), as described in ESM. Tissues (pancreas, liver and white adipose tissue) were dissected, weighed and flash frozen or fixed for immunohistochemistry (anti-CD3, anti-CD68, anti-thioredoxin-interacting protein [TXNIP] and anti-phospho [p][S536]-p65 [1:100; Abcam], anti-ceramide [1:10; Enzo Life Sciences]). Immunoprecipitated insulin receptor (IR) or IRS-2 from liver protein extracts (anti-IRS2 [Cell Signaling], anti-IR [Santa Cruz Biotechnology]; 1 μg) were subjected to Tris-glycine PAGE, immunoblotted with anti-IRS2 or anti-p-Tyr (1:1000; Santa Cruz Biotechnology), visualised by enhanced chemiluminescence and quantified using ImageJ.

Culture media (RPMI-1640), fetal bovine serum, antibiotic–antimycotic solution, L-glutamine, DAPI mounting medium, and PAC and TOP were purchased from Sigma (Sigma-Aldrich, Poznan, Poland). Rabbit monoclonal anti-ALDH1A1 Ab was purchased from Abcam (Abcam, Cambridge, UK), mouse monoclonal anti-COL3A1 was purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA), mouse monoclonal anti-PTPRK Ab, anti-p-Tyr, mouse monoclonal anti-MYOT Ab (B-3), and rabbit polyclonal anti-GADPH Ab were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-P-gp Ab was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Rabbit polyclonal anti-TGFBI was purchased from Atlas Antibodies (Stockholm, Sweden). Mouse anti-Ki67 was purchased from Dako (Glostrup, Denmark). Donkey anti-goat horseradish peroxidase (HRP)-conjugated Ab was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Dallas, TX, USA). The fluorescent MFP488 donkey anti-goat IgG was obtained from MoBiTec (MoBiTec, Molecular Biotechnology, Goettingen, Germany) and fluorescent Alexa Fluor^®488 and Alexa Fluor^®594 Donkey Anti-Rabbit IgG from Jackson ImmunoResearch Laboratories (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK). Western blot reagents (membranes, gels and protein marker) were purchased from Bio-Rad (Bio-Rad Laboratories Ltd., Watford, Hertfirdshire, UK).

Cells were stimulated with 1 μg/ml LPS for the indicated time periods and lysed in weak RIPA buffer (0.5% Triton X-100, 120 mM NaCl, 50 mM Tris-HCl; pH = 7.5) containing phosphatase and protease inhibitors. The whole-cell lysate was sequentially incubated with one of the following antibodies: anti-MyD88, anti-p85α, anti-Nck1, anti-BCAP, or anti-pTyr (Santa Cruz, sc-18182) and shaken overnight at 4°C. Protein A/G-agarose beads (MedChemExpress USA, HY-K0202) were added into the mixture and shaken for a further 2 h, prior to elution with 1 × SDS sample buffer. Prepared samples were further analyzed by immunoblot.

Immunofluorescence analysis was performed on cells growing on microscopic glass slides according to previously established protocol [44 (link)]. For detecting the expression and localisation of PTPRKand/orp-Tyr, cells were fixed and permeabilized with ice-cold acetone/methanol (1:1) for 10 min, rinsed with PBS and blocked in 3% BSA for 30 min at room temperature. After that, cells were incubated for 2 h at room temperature with primary antibodies, anti-PTPRK (mouse monoclonal anti-PTPRK antibody, 1:200, Santa Cruz, CA, USA) and anti-p-Tyr (mouse monoclonal anti-pTyr antibody, 1:200 Santa Cruz, CA, USA). Next, cells were washed with PBS and incubated with the secondary antibody solution for 1 h at room temperature (for PTPRK-Alexa Fluor^®488 Donkey Anti-Rabbit IgG, Jackson ImmunoResearch Laboratories, Cambridgeshire, UK and for p-Tyr-MFP488 Goat Anti-Mouse IgG, Goettingen, Germany). Afterwards, slides were washed with PBS and sealed with DAPI-containing mounting medium. The analysis of immunofluorescence expression was prepared with the use of fluorescence microscope (Zeiss Axio-Imager.Z1, Oberkochen, Germany).