Molecular imager fx pro plus

Samples from two animals were used to validate the proteomics results by one- and two-dimensional western blotting. For this purpose, samples were homogenized and fractionated into membrane, soluble and insoluble proteins, as described above.
Samples for 1D western blot were diluted 1:1 with Laemmli buffer (Bio-Rad, Hercules, CA) and 40 μg of protein were loaded on 10% SDS-PAGE gels. Samples for 2D western blots (40 μg of protein) were loaded in 200 μl of rehydration buffer, and proteins were separated by isoelectric point and then by molecular weight, as previously described30 (link).
Proteins from one or two-dimensional gels were transferred onto a low fluorescence PVDF membrane (Millipore, Billerica, MA) in semi-dry conditions at 15 V for 45 minutes, blocked in 5% fat-free powder milk for 2 hours at room temperature, and incubated overnight with mouse monoclonal anti-rat HSP70 antibody (1:1000, Abcam, Cambridge, MA). Membranes were simultaneously incubated with a rabbit polyclonal anti-rat β-Actin (1:3000, Abcam, Cambridge, MA). For detection, either a goat anti-mouse Cy3-labelled secondary antibody (1:3000, GE, Barcelona, Spain) or a goat anti-rabbit Cy5 (1:3000, GE, Barcelona, Spain) were used, and images were acquired using a Molecular Imager FX Pro-plus (BioRad, Hercules, CA) and analysed with QuantityOne or PDQuest software.

For each Western Blot, ~ 100 gemmules were grown in petri dishes with lake water containing 100 µg/mL ampicillin for 6–13 days at RT. Juveniles were scraped with a razor into 4× SDS-PAGE reducing loading buffer (1 M Tris, pH 7.0, 20% SDS, 20% Glycerol, 0.02% bromophenol blue and 2.5% β-mercaptoethanol), vortexed and boiled at 95 °C for 3 min. Proteins were separated by SDS-PAGE on a 10–12% gel, and transferred to a PVDF membrane (Millipore) at 350 mAmp for 30 min. Membranes were blocked for 1 h at RT in 5% nonfat milk in 1× PBST, pH 7.4 (0.05% Tween 20) and then incubated with affinity purified antibodies (1 mg/mL stocks) against EmVcl (1:3000), EmFAK (1:1000) and EmITGB (1:1500), in blocking solution for 1 h at RT and washed twice in 1× PBST pH 7.4. After 45 min of incubation with the secondary antibody (Alexa488^® Goat Anti-Rabbit IgG Antibody; Life Technologies, 1:1000 dilution) at RT, membranes were washed in 1× PBST pH 7.4 and imaged using the Molecular Imager FX ProPlus (BioRad).

The nuclear extract (2.5 μg) and 105 cpm (0.027 ng) of ³²P labeled oligonucleotides (MWG Biotech, Germany) were incubated in binding buffer (25 mM Hepes, pH 7.6, 5 mM MgCl₂, 34 mM KCl, 2 mM DTT, 2 mM Pefabloc, 2% v/v aprotinin, 40 ng of poly (dI-dC)/μl and 100 ng of bovine serum albumin/μl) for 20 min on ice. Free DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel. Supershift experiments were done with specific antibodies. Gels were blotted to Whatman 3 MM paper, dried under vacuum, exposed to imaging screens for autoradiography and analyzed using a phosphor imaging system (Molecular Imager FX pro plus; Bio-Rad Laboratories GmbH, München, Germany). Gene specific antibodies and probes used are listed in S3 and S4 Tables, respectively.

PCR-products were purified using the NucleoSpin-Extract II kit (Machery Nagel) and plasmids were isolated using the NucleoSpin-Plasmid kit (Machery Nagel). Endonuclease digestions and PCRs were conducted using enzymes purchased from Fermentas, according to manufacturer's instructions. Agarose gel electrophoresis was performed in 1xTAE (40 mM Tris, 1.1 ml/l acidic acid, 1 mM EDTA, 0.5 µg/ml ethidium bromide) buffer.
Radioactive gels were vacuum dried at 80°C, exposed on a phosphor-screen (Kodak), and visualized on a Molecular Imager FX-Pro Plus (BioRad).

Total RNA was isolated from the ventricles by the guanidine thiocyanate CsCl method, and 20-μg samples of RNA were transferred to nylon membranes (Osmonics) for Northern blot analysis as described previously [27 (link),28 (link)]. Full-length rat ANP cDNA probe (a gift from Dr. Peter Davies, Queen’s University, Kingston, Canada), and cDNA probes for rat BNP, SkαA, ß-MHC and 18S were prepared as previously reported [27 (link),28 (link)]. The cDNA probes were labeled, the membranes were hybridized and washed, and exposed with PhosphorImager screens (Amersham Biosciences), which were scanned with Molecular Imager FX Pro Plus and quantified using Quantity One software (Bio-Rad) as previously described [27 (link),28 (link)]. The hybridization signals of specific mRNAs were normalized to that of 18S RNA in each sample.

Purified cofilin or twinfilin (2 µM) was incubated with LIMK1 catalytic domain (2 nM) and ATP (12.5 µM with 0.1 µCi/µL [γ-³²P]ATP) in kinase reaction buffer (defined above) at 30 °C. At 5 min and 10 min incubation, aliquots were removed and quenched with 4x SDS-PAGE loading buffer. Samples were boiled for 5 min and fractionated by 15% acrylamide SDS-PAGE. Gels were Coomassie stained and dried before exposure to a phosphor screen. Phosphor imaging and quantification of radiolabel incorporation were conducted using a BioRad Molecular Imager FX Pro Plus with Image Lab software. Images shown are from the 10 min timepoint. Phosphorylation rates were calculated from the entire time course and normalized to that of wild-type cofilin-1. Michaelis-Menten parameters for cofilin^WT and cofilin^G4F were determined from reactions run at seven substrate concentrations ranging from 50 to 0.5 μM. To calculate absolute rates, a [γ-³²P]ATP standard curve (2.5, 5, and 10 nCi) was imaged alongside the Coomassie-stained gel. Reaction kinetics were calculated using Microsoft Excel 16.75 and GraphPad Prism 9.