Fifty μg of microsomal protein was made up to 50 μL in RB, 50 μL of 2×LB was added and the sample heated at 37°C for 20 min. Proteins were separated by SDS-PAGE (Bio-Rad), and the quantity of recombinant DGAT1 was determined by scanning the immunoblots with a ChemiDoc MP Imaging System (Bio-Rad) and quantifying with Image Lab Software, version 4.1 (Bio-Rad). The specific activity was modified from McFie and Stone (2011) (link) and used a fluorescent 16-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] labeled hexadecanoyl Coenzyme A substrate (Avanti® Polar Lipids Inc., 810705). The TLC plate was developed in the solvent system containing diethyl ether/hexane/methanol/acetic acid (60:40:5:1, v/v/v/v) (Scot J. Stone personal communication). The newly synthesized fluorescent TAG was analyzed with a ChemiDoc MP Imager (Bio-Rad). Chemi-luminescent recombinant DGAT1s and fluorescent TAG was quantitated with the Image Lab software version 4.1 (Bio-Rad).
Imagelab software version 4
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Australia
ImageLab software version 4.1 is a comprehensive image analysis and quantification tool designed for use with Bio-Rad's imaging systems. The software provides a suite of tools for capturing, analyzing, and managing images of gels, blots, and other samples.
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Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (15 mentions)
Ab205718, by Abcam (8 mentions)
Chemidoc mp, by Bio-Rad (7 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Ab181602, by Abcam (5 mentions)
Chemidoc mp imager, by Bio-Rad (5 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Ab9485, by Abcam (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Precast zymography gels, by Thermo Fisher Scientific (3 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp system, by Bio-Rad (3 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Enhanced chemiluminescence substrate, by Thermo Fisher Scientific (3 mentions)
104 protocols using imagelab software version 4
1
Recombinant DGAT1 Activity Assay
2
RT-PCR Analysis of Gene Expression
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Total RNA from the cell lines was isolated using TRIzol reagent (Takara Bio, Inc., Otsu, Japan) and quantified using a NanoDrop ND-2000 (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA), and reversely transcribed into cDNA using the EX Tag kit (TaKaRa, Japan) according to the manufacturer's protocols. RT-PCR was performed utilizing TaKaRa EX Taq DNA Polymerase (Takara Bio, Inc.) under the following conditions: Denaturation at 98°C for 10 sec, annealing at 54–62°C for 30 sec and elongation at 72°C for 1 min for 20–30 cycles. The PCR products were visualized by electrophoresis on a 1.5% agarose gel stained with GelRed™ (Biotium, Inc., Freemont, CA, USA). Images were obtained using the Gel Doc XR+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed using Image Lab™ software version 4.0 (Bio-Rad Laboratories, Inc.). The PCR primers used were: LIMK1, forward 5′-TGAGACAGGTGAGGTGATGG-3′ and reverse 5′-AGGCTGAGTCTTCTCGTCCA-3′; MDR1, forward 5′-ATATCAGCAGCCCACATCAT-3′ and reverse 5′-GAAGCACTGGGATGTCCGGT-3′; and β-actin, forward 5′-CTGGGACGACATGGAGAAAA-3′ and reverse 5′-AAGGAAGGCTGGAAGAGTGC-3′.
3
Immunoblotting of Trypanosoma equiperdum Antigen
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Onderstepoort Veterinary Institute strain of T. equiperdum antigen was purified from rat blood as described in Ref. (9 (link)). Animal experimentation was done according to Italian national law (Legislative Decree 26/2014) (10 ) and Directive 2010/63/EU on the protection of animals used for scientific purposes (11 ). Ethical approval was obtained from the Italian Ministry of Health (Protocol no. 114/2014 PR of 19.12.2014, ex Lgs. D. 26/2014, art. 31).
Immunoblotting (cIB) was performed according to Ref. (9 (link)), using the purified OVI T.e. antigen and NuPage® 12% Bis-Tris pre-cast gels (Life Technologies, Paisley, UK) at 200 V. OVI T.e. proteins were then transferred onto a nitrocellulose membrane. After blocking with skim milk, membranes were cut into strips, which were incubated with sera diluted 1:10 and then with a monoclonal antibody anti-horse IgG-HRP-conjugate (MAb IZSA&M, Italy).
Antigen-antibody reactions were visualized by adding the AmershamTM ECL SelectTM Western Blotting Detection Reagent (GE Healthcare, Uppsala, Sweden). Images were acquired using the ChemiDoc MP (Bio-Rad) and the Image Lab Software, version 4.0 (Bio-Rad); detection time ranged from 1 to 5 s, for both positive and negative sera. As molecular weight marker, BenchMarkTM Prestained Protein Ladder (Life Technologies) was used.
Immunoblotting (cIB) was performed according to Ref. (9 (link)), using the purified OVI T.e. antigen and NuPage® 12% Bis-Tris pre-cast gels (Life Technologies, Paisley, UK) at 200 V. OVI T.e. proteins were then transferred onto a nitrocellulose membrane. After blocking with skim milk, membranes were cut into strips, which were incubated with sera diluted 1:10 and then with a monoclonal antibody anti-horse IgG-HRP-conjugate (MAb IZSA&M, Italy).
Antigen-antibody reactions were visualized by adding the AmershamTM ECL SelectTM Western Blotting Detection Reagent (GE Healthcare, Uppsala, Sweden). Images were acquired using the ChemiDoc MP (Bio-Rad) and the Image Lab Software, version 4.0 (Bio-Rad); detection time ranged from 1 to 5 s, for both positive and negative sera. As molecular weight marker, BenchMarkTM Prestained Protein Ladder (Life Technologies) was used.
4
Western Blot Protein Quantification
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Protein separation was carried out on 50 µg samples of total protein in a 12% tris-glycine gel (Thermo Scientific, Milan, Italy). Protein was transferred onto nitrocellulose membrane (Macherrey-Nagel, Düren, Germany) overnight at 12 mV. Membranes were blocked with a buffer containing 5% fat-free milk and then incubated overnight at 4°C with a mouse anti-TP primary antibody (Thermo Scientific, Milan, Italy) at a final concentration of 2 µg/ml. Membranes were washed three times and incubated with an anti-mouse peroxidase-conjugated secondary antibody (Sigma Aldrich, Milan, Italy). Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) on a ChemiDoc MP System and quantified by Image Lab software version 4.0 (Bio-Rad Laboratories, Hercules, CA, USA). Band intensities were expressed relative to total protein and/or to the intensity of GAPDH detected on the same membrane following stripping with Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific, Pittsburgh, PA, USA) and overnight incubation with GAPDH antibody at 4°C at a 1∶1000 dilution (Abcam, Cambridge, UK). Each assay was conducted in technical triplicate.
5
SDS-PAGE Analysis of Bacterial Lipoproteins
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed according to the method of Laemmli [42 (link)], as follows. Each culture supernatant or purified protein was mixed with reduced Laemmli sample buffer containing dithiothreitol (DTT; final concentration 200 mM) and incubated at 100 °C for 3 min. Each sample was loaded on an Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-Rad, Hercules, CA, USA) and subjected to electrophoretic separation. Chemifluorescent signals were captured using a Chemi Doc MP Imaging system (Bio-Rad). Precision Plus Protein Unstained Standards (Bio-Rad) were used as molecular weight markers. The protein bands were analyzed using Image Lab software version 4.0 (Bio-rad). The protein level of mature BLP in each culture supernatant was calculated from the intensity of the band at the position of mature BLP (19 kDa), using the serial diluent of purified wild-type BLP (quantified by DC-protein assay kit (Bio-Rad)) as the standard. Non-reducing SDS-PAGE was performed in the same way as above, but using DTT-free Laemmli sample buffer instead of the reduced buffer.
6
Aortic MMP2 and MMP9 Activity Assay
7
Western Blot Analysis of Adipogenic Markers
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Cells were washed and harvested with ice-cold DPBS. Lysates were prepared using radioimmunoprecipitation cell lysis buffer containing 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxylcholic acid, 10% NP-40 and 10 mM EDTA. Protein concentration was measured using a Bicinchoninic Acid Protein assay kit (Thermo Fisher Scientific Inc.). Cell lysates (30 µg) were separated by 4–20% Criterion™ TGX™ precast gel (Bio-Rad Laboratories, Inc.) electrophoresis and subsequently transferred onto polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Chalfont, UK). Membranes were blocked with 5% skimmed milk and incubated overnight at 4°C with C/EBPα, PPARγ and β-actin primary antibodies at a dilution of 1:1,000. After 1 h incubation at room temperature with HRP-conjugated anti-mouse for or anti-rabbit secondary antibodies (1:3,000), protein bands were detected with an Enhanced Chemiluminescence assay kit (Thermo Fisher Scientific, Inc.). Images were captured using a ChemiDoc™ XRS+ image analyzer (Bio-Rad Laboratories, Inc.). Relative density of C/EBPα and PPARγ were normalized to β-actin and quantified using Image Lab™ software version 4.0 (Bio-Rad Laboratories, Inc.).
8
Immunoblotting Analysis of Melanoma Proteins
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To determine protein expression, cultured B16-F0 cells were analyzed after 24 h of in vitro treatment. Melanomas generated in vivo by B16-F0 subcutaneous inoculation were also studied. Immunobloting was performed as described previously (Hapon et al., 2014 ). To quantify and statistically compares the protein expression, cultures were performed by triplicated and three melanomas group were studied. Primary antibodies used were: cyclin-dependent kinase inhibitor p21cip1 (Termofisher AHZ0422), proliferating cell nuclear antigen (PCNA, Termofisher MA511358), caspase 3 (Casp3, Invitrogen, 74T2), cleaved poly (ADP-ribose) polymerase (cPARP, Termofisher 44698G), alpha tubulin (TUBA1A, Termofisher 322500). Peroxidase conjugate secondary antibodies used were: biotin conjugated antimouse and antirabbit (Jackson 115-035-003 and 711-065-152). Blots were developed using a ChemiDoc XRS + System (Bio-Rad, Laboratories) and band densitometric analysis was performed using Image Lab Software version 4.0 from Bio-Rad Laboratories.
9
Western Blot Analysis of Apoptosis Regulators
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The treated cells (5–6×106) were washed in PBS and then lysed in 2X Laemmli buffer. Protein concentrations were determined using the BCA assay kit.
Following boiling for 5 min, equivalent quantities of protein (30–40 µg) were separated on 8–12% SDS-polyacrylamide gels and then transferred onto a nitrocellulose membrane (0.45 µM). The membranes were blocked with 5% BSA in TBS/Tween20 (0.05% v/v) for 1 h, followed by incubation at 4°C overnight with primary antibodies against Bcl-2 (1:500 dilution), survivin (1:870 dilution), Akt (1:1,000 dilution), p-Akt (Thr308; 1:1,000 dilution) and p-Akt (Ser473; 1:1,000 dilution). GADPH (1:1,000 dilution) served as a control. The membranes were washed three times with TBST and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:10,000 dilution). The protein bands were visualized using enhanced chemiluminescence on a ChemiDoc™ XRS+ system with Image Lab™ software version 4.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The quantification of protein levels was performed by Gel-Pro Analyzer (version 4.0) and the integrated option density was used as the quantity for comparison.
Following boiling for 5 min, equivalent quantities of protein (30–40 µg) were separated on 8–12% SDS-polyacrylamide gels and then transferred onto a nitrocellulose membrane (0.45 µM). The membranes were blocked with 5% BSA in TBS/Tween20 (0.05% v/v) for 1 h, followed by incubation at 4°C overnight with primary antibodies against Bcl-2 (1:500 dilution), survivin (1:870 dilution), Akt (1:1,000 dilution), p-Akt (Thr308; 1:1,000 dilution) and p-Akt (Ser473; 1:1,000 dilution). GADPH (1:1,000 dilution) served as a control. The membranes were washed three times with TBST and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:10,000 dilution). The protein bands were visualized using enhanced chemiluminescence on a ChemiDoc™ XRS+ system with Image Lab™ software version 4.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The quantification of protein levels was performed by Gel-Pro Analyzer (version 4.0) and the integrated option density was used as the quantity for comparison.
10
Liver Protein Expression Analysis
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Liver samples were homogenized in tissue lysis buffer (RIPA: protease inhibitor: phosphorylated protease inhibitor = 100:1:1) and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected and stored at −80 °C for later use. The protein concentration of the sample was measured according to the BCA kit procedure. Proteins were separated by electrophoresis and then transferred to a polyvinylidene fluoride membrane, which was incubated at 4 °C overnight with specific antibodies (LKB1, AMPK, p-AMPK, TSC2, p-TSC2, mTOR, p-Mtor, PPARγ, GAPDH, β-ACTIN) (CST, United States), followed by incubation with secondary antibodies (anti-rabbit IgG, HRP-linked antibody) (CST, Danvers, MA, USA) for 1.5 h at room temperature. Next, the membrane was washed three times with 1 × TBST solution for 10 min each time. The membranes were submerged in the developer solution after absorbing the water with filter paper and then placed in a gel imaging scanning system for development. The intensity of the gray bands was quantified using Image Lab software (version 4.0, Bio-Rad, Hercules, CA, USA).
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