Immediately after excision from mice, some of the tumors were fixed in 5% formalin overnight after which they were dehydrated in ethanol and embedded in paraffin. Tumor sections (5–6μm) were deparafinized, and immunolabeling was performed as described previously (21 (link)). Cells from dissociated HMLEHRASV12 xenografts (both transplantable and non-transplantable) were plated in six well plates and allowed to propagate for 48 hrs. Cells were fixed with 4% paraformaldehyde in 1× PBS for 30 min, washed, and incubated with blocking buffer (TBS pH 7.8, 5% normal goat serum, 1% BSA and 1% triton X-100) for 1hr. The following antibodies were used in our experiments: UCP1 (ab10983, Abcam), UCP2 (AB3226, Millipore, Billerica, MA); and UCP3 (ab3477, Abcam).
Ab3477
Manufactured by Abcam
Sourced in United Kingdom
Ab3477 is a lab equipment product offered by Abcam. It serves a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab10983, by Abcam (2 mentions)
Ab3226, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Ab4074, by Abcam (1 mentions)
Ab97931, by Abcam (1 mentions)
Cellytic mt cell lysis reagent, by Merck Group (1 mentions)
Membrane blocking reagent, by GE Healthcare (1 mentions)
Ecl select western blotting reagent, by GE Healthcare (1 mentions)
Mitochondria isolation kit, by Thermo Fisher Scientific (1 mentions)
Ab23841, by Abcam (1 mentions)
Ab10527, by Merck Group (1 mentions)
Luminata classico western hrp substrate, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
G box gel documentation system, by Syngene (1 mentions)
Protease and phosphatase inhibitor cocktail mix, by Merck Group (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Ab14744, by Abcam (1 mentions)
Prdm16, by Santa Cruz Biotechnology (1 mentions)
6 protocols using ab3477
1
Immunolabeling of Dissociated Xenograft Cells
2
Western Blot Analysis of Mitochondrial Proteins
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Each tissue was homogenized in micro tubes with lysis buffer (CelLytic™ MT cell lysis Reagent: SIGMA-Aldrich, Japan) containing protease inhibitor (SIGMA-Aldrich Japan) and 0.2% SDS. The protein concentration was measured by the Bradford method. A 50 μg aliquot of protein was separated by SDS-PAGE using 4-12% Bis-Tris gel and then transferred onto polyvinylidene difluoride membranes (Life Technology, USA). The membranes were blocked with membrane blocking reagent (GE Healthcare, Buckinghamshire, UK) for 1 hour, and then incubated for 2 hours with rabbit polyclonal primary antibodies against either UCP1 (1:2500; ab1983, Abcam, Cambridge, UK), UCP2 (1:2500; ab97931, Abcam), UCP3 (1:2000; ab3477, Abcam), CPT-2 (1:500; sc-20671, Santa Cruz, Santa Cruz, USA), MCAD (1:1000; sc-98926, Santa Cruz) or α-tubulin (1:2000; ab4074, Abcam). After the primary antibody reaction, the membranes were incubated for 1 hour with the appropriate horseradish peroxidase-conjugated secondary antibody (1:100000). Immunoreactivity was detected by chemiluminescence using the ECL select™ Western Blotting Reagent (GE Healthcare, Buckinghamshire, UK). Fluorescence band images were analyzed using Just TLC (Liponics, Tokyo, Japan) analysis software. Each value was normalized relative to α-tubulin.
3
Mitochondrial Protein Analysis in BAT
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Interscapular BAT and GM from male TRPV1 WT and KO mice and WT mice administered with AMG9810 or vehicle were immediately dissected and snap frozen in liquid nitrogen for mitochondrial protein extraction using a Mitochondria Isolation Kit (Thermo Scientific) according to the manufacturer’s instructions. Equal amounts of protein (15–30 μg) were separated by SDS-PAGE under reducing conditions in 10% polyacrylamide gels. The proteins were transferred to PVDF membranes (EMD Millipore, Feltham, United Kingdom), blocked in 5% nonfat dried milk (1 h at room temperature), and incubated with primary antibody for UCP1 and UCP3 (1:1000 ab23841 and ab3477; Abcam, Cambridge, United Kingdom) at 4°C for 12 h. The voltage-dependent anion-selective channel (VDAC) was used as a loading control (1:1000 AB10527; EMD Millipore). The membranes were exposed under ECL (Luminata Classico Western HRP substrate; EMD Millipore) in a G-BOX Gel Documentation System (Syngene, Cambridge, United Kingdom) using the manufacturer’s software (Syngene 2D gel imaging software; Syngene). Samples were quantified densitometrically using ImageJ (National Institutes of Health, Bethesda, MD, USA).
4
Protein Extraction and Immunoblotting from Frozen Heart Tissue
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Frozen heart tissue was homogenized in ice-cold buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, and 1% NP-40, and supplemented with protease and phosphatase inhibitor cocktail mix (Sigma-Aldrich). The homogenate was centrifuged at 1000 g for 10 min at 4°C. The resulting supernatant was collected for protein concentration determination and used for immunoblotting in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein concentration was measured by Bradford assay. The primary antibodies (Akt, pAkt, ERK1/2, pERK1/2, all from Cell Signaling Technology, Danvers, MA) were polyclonal rabbit antibodies. Mouse monoclonal anti-COX IV antibody (clone 20E8C12, ab14744, mitochondrial loading control), polyclonal rabbit anti-COXIV isoform 2 (ab70112), and polyclonal rabbit anti-UCP3 (ab3477) were purchased from Abcam Inc. (Cambridge, MA, USA). Polyclonal goat anti-ANT (N-19) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Immunoreactivity was visualized by horseradish peroxidase-conjugated antibodies using a peroxidase-based chemiluminescence detection kit (ECL) (PerkinElmer, Woodbridge, Ontario, Canada). The intensity of the bands was quantified by two individuals (PHL, EL) independent of each other using ImageJ software (http://rsbweb.nih.gov/ij/ ).
5
Protein Analysis of Xenograft Samples
6
UCP3 Expression via Western Blotting
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The UCP3 expression was assessed by western blotting analysis, and the samples were normalised to VDAC protein. Mitochondrial protein extraction was blocked with PBS-5% fat-free dried milk at room temperature for 1h, and incubated overnight at 4-8℃ with anti-UCP3 (1:1000, ab-3477,Abcam), and anti-VDAC (1:1000, ab-61273，Abcam) antibodies. [20] At fifteen days after birth,the tails of the mice were collectedand digested overnight at 55˚C by tissue cracking buffer containing protease K. DNA was extracted from the mice tailsas previously described. PCR were employed to genotype mice identification. The following primer sequences were used for detection: 5'CCT CCA CTC ATG ATC TAT AGA TC3', 5'GCA CTG CGG CCT GTT TTG3', and 5' ACC CTC TGT GCG CAC CAT AGT CA3'. The PCR products were analysed by agarose electrophoresis.
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