Halt protease phosphatase inhibitor cocktail

Experiments were conducted to compare a single tissue solubilization step using an acid labile surfactant to approaches for tissue decellularization. The single step method used the acid-labile surfactant Protease MAX surfactant with heating (MAX). Two tissue decellularization methods incorporated sequential decellularization with solubilization of the residual pellet with MAX. First tissue decellularization approach used 0.4% SDS + HALT protease/phosphatase inhibitor cocktail (Thermo Fisher) followed by solubilization of residual “ECM” pellet using MAX (SDS.MAX). Second tissue decellularization approach used sequential decellularization with 25mM NH4OH/ 0.5%TritonX-100 (TX) followed by solubilization of residual “ECM” pellet using MAX (TX.MAX). As described in Hobeika L et al. [36 (link)], the tryptic peptides were analyzed using a LC-MS Orbitrap ELITE approach with peptide assignments using a Mascot/Sequest search strategy. Scaffold4 was used to set false discovery rate (FDR) control. Finally, we obtained a label-free quantified data of identified proteins (Supplementary File 1). Please see more details about the experimental procedures in “Supplementary File 2”. We analyzed the data for comparing statistical methods with MVs in the presence of heterogeneity.

CD8⁺ T cells were lysed with RIPA buffer (50mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% Sodium deoxycholate, and 150mM NaCl) with addition of Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific, Waltham, Massachusetts, U.S.A.) and protein concentration was determined using the Bio-Rad protein assay (Hercules, California, U.S.A.). Proteins were resuspended in LDS Sample Buffer, loaded (15 μg/lane) onto NuPAGE Bis-Tris gels, and transferred to polyvinylidene difluoride (PVDF) membranes using iBlot2 system (LifeTechnologies, Grand Island, New York, U.S.A.). Proteins were detected using rabbit anti-S6K, phospho-S6K, AMPKα, phospho-AMPKα, Acetyl-CoA carboxylase (ACC), phospho-ACC, cyclin E, cdk2, phospho-Rb (Ser780), tubulin, and anti-rabbit IgG HRP-linked antibody from Cell Signaling Technology (Danvers, Massachusetts, U.S.A.). Primary antibodies were used at a 1:1,000 dilution whereas secondary antibody was used at a 1:50,000 dilution. PVDF membranes were developed using ECL Western Blotting Substrate (Thermo Scientific, Waltham, Massachusetts, U.S.A.) and exposed to Amersham hyperfilm ECL (GE Healthcare Life Sciences, Piscataway, New Jersey, U.S.A.)

Cell lysates were prepared in RIPA buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM disodium EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1% triton-X100) plus 1 mM phenylmethylsulphonyl fluoride and Halt protease/phosphatase inhibitor cocktail (Thermo Scientific). Protein concentration was measured by Bradford method (Thermo Scientific). 50 μg protein was separated by 4–15% sodium dodecyl sulfate polyacrylamide gels (BioRad Hercules), transferred to PVDF membranes (GE Water and Process Technologies), blocked in Tris-buffered saline (pH 7.4) plus 0.1% Tween-20 and 5% skim milk, and incubated overnight at 4° C with 1:1000 diluted phospho- and/or total antibodies against indicated proteins (Cell Signaling) plus anti-mouse β-actin (Santa Cruz Biotechnology). Membranes were incubated with horse radish peroxide-conjugated antibodies, 1 h. Proteins were detected by enhanced chemiluminescence (Pierce). Band quantification and normalization to total protein was by ImageJ software (22 (link)). Data show means of 3 individual blots with comparisons only made between like blots from the same gels.