Image station 4000mm pro

Cells were harvested and lysed with RIPA buffer (50 mM Tris–Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% NP-40) containing protease inhibitor cocktail (Abcam, Cat No. ab65621). The lysates were centrifuged at 12,000 rpm for 30 min at 4°C. The protein concentrations of supernatants were determined by BCA protein assay (Thermo Scientific, Rockford, IL, USA). The protein extract of each sample (25 μg) was electrophoresed on 10%–12% polyacrylamide gel with sodium dodecyl sulfate and then transferred onto nitrocellulose membrane (Millipore A) at 100 V for 1.5 h. After being blocked with 3% BSA in TBST (TBS-1% Tween 20) for 1 h, the membranes were incubated with 1:1,000 primary antibodies (purchased from CST, Danvers, MA, USA) of TRIM28 (Cat No. #4124), VIM (Cat No. 5741S), CCND1 (Cat No. 55506s), PCNA (Cat No. D3H8P), Bcl-2 (Cat No. 5071S), CDH1 (Cat No. 3195s), CDH2 (Cat No. 13116s), Snail (Cat No. 3879S), or PARP(Cat No. 46D11) overnight at 4°C, respectively, then washed and further incubated with secondary horseradish peroxidase-conjugated anti-rabbit IgG. Finally, protein bands were detected by developing the blots with Immobilon ECL Ultra Western HRP Substrate (Millipore, Cat No. WBULS0500) and pictured on Image Station 4000MM Pro (Carestream, Canada). ImageJ was used to quantify protein levels relative to load control β-actin.

Supernatants obtained from stimulated splenocytes culture were analyzed with proteome profiler arrays (Proteome Profiler Mouse Cytokine Array Kit, Panel A; R&D Systems, Minneapolis, MN, USA) according to the enclosed instructions. Pixel densities on X-ray film were collected using a multifunctional scanning device Samsung SLC460 (Samsung, Suwon, Korea) or Image Station 4000 MM PRO (Carestream Health, Rochester, NY, USA), after which image analysis was performed (ImageJ 1.48v). For each spot, the final level of optical density was determined as a factor acquired by subtracting the background optical level and dividing by values obtained from the non-treated mice (named as day 0).

Protein extracts were prepared, separated, and transferred to membranes as previously described [66 (link)]. Membranes were blocked in 5 % nonfat dry milk (w/v) in Tris-buffered saline (pH 7.0) containing 0.1 % Triton-100 (TBS-T) for 1 h at room temperature. Membranes were incubated at 4 °C overnight with the following: 1:2,500 rabbit anti-phospho-p38 MAPK antibody (Cayman Chemical), 1:2,000 rabbit anti-phospho-MK2 antibody (Abgent), or with 1:10,000 rabbit anti-GAPDH antibody (Abcam) in 5 % non-fat dry milk in TBS-T. Anti-phospho-p38 MAPK antibody and anti-phospho-MK2 antibody were raised against mammalian proteins; peptide sequences used to produce these monoclonal antibodies have greater than 90 % identity to their counterparts in A. stephensi. Membranes were washed three times for 5 min each in 1X TBS-T and incubated with a 1:20,000 dilution of HRP-conjugated goat anti-rabbit (Fab’)2 fragment (Cell Signaling Technology) at 4 °C overnight. To reveal antibody-bound proteins, membranes were incubated with SuperSignal West Dura chemiluminescent reagent for 5 min and visualized using the Kodak Image Station 4000MM Pro and Carestream Molecular Imaging software (Carestream Health). Levels of phospho-proteins in each treatment were first normalized to total protein levels as determined by GAPDH and then to the appropriate control group.

Cells were lysed by suspension (1 h at 4°C) in the lysis buffer A containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 10% glycerol, 1 mM EDTA, 150 mM NaCl and protease and phosphatase inhibitor cocktails. Protein concentration was determined by Bradford method. Proteins (20-30 μg) were subjected to 9% or 11% SDS-PAGE, blotted on Immobilon-P membranes (Sigma-Aldrich), processed in western blot with the indicated antibodies and developed using an enhanced chemiluminescent detection system (ECL). Immunostained bands were quantified by means of a Kodak-Image-Station 4000MM-PRO and analysis with Carestream Molecular Imaging software (New-Haven, CT).