Pierce ecl western blotting detection system

Liver samples were homogenized in tissue lysis buffer using a TissueLyser II instrument (Qiagen, Hilden, Germany). The tissue lysis buffer contained 1% Triton X-100, 150 mM NaCl and 20 mM Tris-HCl pH 7.5 and was supplemented with protease and phosphatase inhibitors. Lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with the following primary antibodies: STAT3, p-STAT3 (Tyr705), p-RB (S807/811), p-CDK2 (T160), cyclin E1, and GAPDH (all from Cell Signaling Technology, Danvers, MA); β-tubulin and CDK1 (both from Abcam, Cambridge, MA); CD81 (St. John’s Laboratory, London, UK); and cyclin D1 (Santa Cruz Biotechnology, Dallas, TX). Second stage goat-anti-rabbit-HRP was obtained from Jackson ImmunoResearch Laboratories Inc. Antibody binding was visualized using the Pierce™ ECL western blotting detection system (Thermo Fisher Scientific, Waltham, MA). Densitometric analyses were performed using the ImageJ software (https://imagej.nih.gov/ij/).

SDS-PAGE and immunoblotting were performed according to standard protocols using antibodies reactive against the c-Myc epitope for wild-type and mutant PLCγ₂. Immunoreactive proteins were visualized using the Pierce ECL Western blotting detection system (#32106, ThermoFisher Scientific). Samples to be analyzed by Western blotting were taken, quasi as a fourth replicate, from the same plate as and immediately adjacent to the samples taken in triplicate for functional analysis. Using this protocol and paying meticulous attention to experimental detail, we have not experienced variations in gel loading of these samples. All experiments were performed at least three times. Similar results and identical trends were obtained each time. Data from representative experiments are shown as means ± S.E. of triplicate determinations. In Figure 1, 2, 3B, 4, 8, 9A, 9C, 10B, 11, and 13, the data were fitted by nonlinear least squares curve fitting to three- or four parameter dose-response equations using GraphPad Prism, version 5.04. In certain cases, the global curve fitting procedure contained in Prism was used to determine whether the best fit values of selected parameters differed between data sets. The simpler model was selected unless the extra sum of squares F-test had a p value of less than 0.05.