Cellytic mt mammalian tissue lysis extraction reagent

Lung tissues from infected C57Bl/6 mice were collected and homogenized using 200ul of Sigma CelLytic MT Mammalian Tissue Lysis/Extraction reagent per 40mg lung tissue. Following lysis membranes were pelleted at 14,000xg for 10 minutes and samples were stored at -80°C. 30μg of cell lysate was loaded on a denaturing 12% SDS polyacrylamide gel and detected with TGFβ1 antibody (Ebioscience clone A75-2), membranes were stripped and probed for β-actin.

Mice were sacrificed 4 and 24 h after CCI-induced TBI, and the brains were removed. Each brain was separated into two parts: the lesioned hemisphere and the contralateral intact hemisphere. Brain tissue was collected and stored separately in liquid nitrogen. Proteins were extracted from the injured cerebral hemisphere and the intact contralateral hemisphere, using the CelLytic MT mammalian tissue lysis/extraction reagent (Sigma-Aldrich, C3228, St. Louis, MO, USA). The antibodies used to detect the blot were rabbit monoclonal anti-alpha Fodrin (EPR3017)-SBDP150 (Abcam, ab75755, Cambridge, UK), monoclonal anti-GFAP (Millipore, MAB360, Billerica, MA, USA), and purified mouse monoclonal antibody Beclin-1 [BD Biosciences, 612113, Fanklin Lakes, NJ, USA; Santa Cruz Biotechnology, Inc., sc-9888, Dallas, TX, USA]. Mouse monoclonal anti-β-actin (Sigma-Aldrich, A5441, St. Louis, MO, USA) served as an internal control. Cell lysates were resolved with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted with the antibodies mentioned above, and incubated with the corresponding secondary antibodies. Proteins were visualized by following the manufacturer’s instructions (Pierce ECL Western blotting substrate, Thermo Scientific, Waltham, MA, USA). The experimenter was blinded to the samples when the protein expression was quantified.

Hearts or coronary arterioles were homogenized in lysis buffer (Cellytic MT Mammalian Tissue/ Lysis/extraction Reagent; Sigma). Protein concentrations were measured using a BCA Protein Assay Kit (Pierce) and equal amounts of protein (10, 20, or 40 μg) separated by SDS-PAGE and transferred onto nitrocellulose membranes. Protein expression was detected using the appropriate primary antibody: TNF-α (1:500; R&D Systems), IL-6 (1:500; Abcam, Inc.), SOD2 (1:1,000; EMD Chemical), eNOS (1:100; Santa Cruz Biotechnology), p-eNOS (1:100; Santa Cruz Biotechnology), β-actin (1:2,000; Abcam Inc.), and corresponding secondary antibodies (1:1,000~2,000 dilution). Signals were enhanced by chemiluminescence (ECL; Amersham) and visualized with a Fuji LAS3000 densitometer. The density of protein bands obtained from the images was analyzed using Multigauge software (Fuji film). The relative densities were calculated and normalized to those of the corresponding internal reference β-actin, and then normalized to the corresponding WT control mice, which was set to a value of 1.0.

The prostate and bladder tissues of rats injected with formalin or saline (n = 6 per group) were homogenized using cold CelLytic™ MT Mammalian Tissue Lysis/Extraction Reagent (sigma) containing 2 mM sodium orthovanadate, 1 mM PMSF and protein cocktail inhibitor (1X, Sigma). The homogenate was centrifuged at 10,000 rpm for 10 min and the resulting supernatants were stored at -80 °C until assayed. 28 proteins including interleukins IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A and IL-18; CXC chemokines (CXCL1, CXCL2, CXCL5 and CXCL10), CC chemokines (CCL2, CCL3, CCL5); Growth factors NGF, BDNF, VEGF and G-CSF, other inflammatory mediators such as eotaxin, leptin, IFN-γ and TNFα levels were determined on a Luminex 100 using a MILLIPLEX MAP Rat Cytokine/Chemokine Panel (Millipore, Billerica, MA). Levels of NGF and BDNF were determined using individual ELISA kits procured from Promega, USA. Protein estimation was done by BCA Protein Assay Kit (Pierce, Rockford, Illinois) to standardize the chemokine concentrations relative to tissue protein levels, which are expressed as pg/mg of total protein [12 (link)].

Mouse tissue (frozen whole skin and muscle samples) and 3D-keratinocytes were homogenized and sonicated in CelLytic MT Mammalian Tissue Lysis/Extraction Reagent (Sigma), and then centrifuged at 16,000g for 10 min at 4°C. The supernatants were harvested, and equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies to KRT5 (Abcam, Cambridge, UK), KRT10 (Abcam), KRT14 (Santa Cruz Biotechnology, Dallas, TX), desmoglein 1 (DSG1; GeneTex, San Antonio, TX), DSG2 (Abcam), desmocollin 3 (DSC3; Santa Cruz Biotechnology), α-tubulin (Cell Signaling), Akt (Cell Signaling), phospho-Akt (Ser473) (Cell Signaling), p70S6K (Cell Signaling), and phospho-p70S6K (Thr389) (Cell Signaling). After washing the membranes with 0.1% Tween 20 in PBS, they were exposed to a horseradish peroxidase-linked anti-rabbit IgG antibody (Cell Signaling). Bands were visualized with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and their intensities were quantified using the program Multi Gauge (Fujifilm, Tokyo, Japan).