Las 1000 charge coupled device camera

Western blot was performed as described in a previous publication [37 (link)]. Primary antibodies used were anti-PKCδ (1:500, Santa Cruz), anti-Smac (1:500, Santa Cruz), anti-Actin (1:2000, MP Biomedicals), anti-HSV (1:1000, Novagen), anti-GST (1:2000, GE Healthcare) and anti-GFP (1:1000, Invitrogen). Secondary horseradish peroxidase-labeled antibodies used were from GE Healthcare and Dako. For the chemiluminescent reaction, Supersignal Substrate (Thermo Scientific) was used according to manufacturer’s instructions. Chemiluminescence was detected with a LAS-1000 charge-coupled device camera (Fujifilm) and Image Reader LAS-1000 Pro v2.6 software (Fujifilm). Image quantifications were performed using ImageJ 1.48v and by normalizing band intensities to input fractions and control.

For western blotting, MCs cultures were lysed in a buffer containing 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS). The total protein was quantified using a protein assay kit (Bio-Rad). Total cell protein (50 mg) was resolved on 8-10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes, which were then blocked with fat-free milk and probed with specific antibodies against b1 adrenergic receptor (Santa Cruz Biotechnology). Images of the blots were acquired with a LAS-1000 Charge Coupled Device camera (Fujifilm).
For quantitative RT-PCR analysis, MCs were lysed in TRI Reagent (Ambion), and RNA was extracted according to manufacturer's instructions. Complementary-DNA was synthesized from 2 μg of total RNA by reverse transcription (RNA PCR Core Kit, Applied Biosystems). Quantitative PCR was carried out in a Light Cycler 2.0 using a SYBR Green Kit (Roche Diagnostics GmbH) and specific primers sets for human Snail, E-cadherin, fibronectin, pro-collagen-I, α-SMA and histone H3. Samples were normalized with respect to the value obtained for H3. All experiments were repeated at least three times. For RT-PCR determinations in each experiment it was assigned a value of one to a control omentum (C-) from which the relative values of the remaining groups were calculated. The primer sets used are depicted in Table S1.