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5 mmol/L Tween 20, pH 7.5) and then were incubated with primary antibodies overnight. The following antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA): Tasisulam Rheb (#4935), S6 (#2317), phosphor-S6 (S235/236) (#2211), phosphor-S6 (S240/244) (#2215), 4E-BP1 (#9452), phosphor-4E-BP1 (T37/46) (#9459), phosphor-4E-BP1(S65) (#9451), p70S6K (#9202), phospho-p70S6K (Thr389) (#9206), total Akt (#9272), phospho-Akt (Thr308) (#9275), phospho-Akt (Ser473) (#9271), extracellular signal�Cregulated kinase (ERK; #9102), phospho-ERK (Tyr202/Tyr204) (#9101), p38 (#9212), and phospho-p38 (Thr180/Tyr182) (#9211). Pan-actin antibody (#MS-1295-P0) and horseradish peroxidase�Clinked secondary antibodies (#31460 and #31430) were purchased from Thermo Scientific (Rockford, IL). H&E and Masson��s staining and immunofluorescence were performed as described previously.7, 15?and?16 Briefly, heart Inhibitor Library samples were first washed with cold PBS and then were fixed in 4% paraformaldehyde at 4��C. The samples were processed successively by i) a 30-minute wash in PBS at 4��C; ii) 15-minute incubations in 30%, 50%, 75%, and 85% ethanol and then two 10-minute incubations in 95% and 100% ethanol at room temperature; iii) three 10-minute incubations in xylene at room temperature; iv) a 20-minute incubation in paraffin/xylene (dilution 1:1) at 65��C; and v)?three 30-minute incubations in fresh paraffin at 65��C. The processed samples were then embedded in paraffin and sectioned (6 ��m thick), and the sections were stained. Immunofluorescence staining was performed using anti�Ccardiac troponin T antibody and anti�Cwheat germ agglutinin antibody at 4��C overnight. Fluorescence microscopy images were obtained using a research fluorescence microscope (Olympus America Inc., Center Valley, PA) equipped with a digital camera. Images were collected learn more and recorded using Photoshop version 5.0 (Adobe Systems Inc., San Jose, CA) on an IBM R52 computer (IBM, Armonk, NY). TUNEL assay was performed as previously reported.7?and?15 Briefly, the sections were treated with 20 ��g/mL of proteinase K and were incubated with terminal deoxynucleotidyl transferase and biotinylated deoxyuridine triphosphate. Data are presented as means �� SEM values. For comparisons between two groups, statistical significance was determined using the unpaired two-tailed Student's t-test. A P