Differentially abundant proteins of interest were examined by immunoblotting of pools of protein extracts from all experimental groups and controls from Parts I and II. Protein (10 µg per pool) was fractionated on 12% Criterion TGX Stain-Free gels (Bio-Rad). Gel images were acquired with the Chemidoc system (Bio-Rad) to document equal protein loading among samples. Then, proteins were electrotransferred onto nitrocellulose membranes and probed with following primary antibodies: anti-ATP5F1A (1:1000; #ab151229, AbCam), anti-SDHB [21A11AE7] (1:500; #ab14714, AbCam), anti-PGA3 (1:1000; #PA5-49728, Thermo Fisher Scientific), anti-pepsinogen II/PGC (1:500; #ab135862, AbCam), anti-PDIA3 (internal region; 1:1000; #ABIN3187755, Antibodies-online; Aachen, Germany), anti-GSTP1 (1:1000; #PA5-29558, Thermo Fisher Scientific), and anti-PSME1 (1:1000; #ab14714, AbCam). Primary antibodies were detected with HRP-conjugated goat anti-rabbit and anti-mouse IgG Fc fragment antibodies (1:10,000 dilution; Bethyl Laboratories, Montgomery (TX), USA) and Clarity Western ECL Substrate (Bio-Rad). Blots were imaged with the Chemidoc system.
Ab14714
Manufactured by Abcam
Sourced in United Kingdom, Germany, United States
Ab14714 is a primary antibody that targets the protein Troponin C. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab14705, by Abcam (9 mentions)
Ab110242, by Abcam (9 mentions)
Ab14745, by Abcam (8 mentions)
Ab14748, by Abcam (7 mentions)
Ab14734, by Abcam (5 mentions)
Ab14715, by Abcam (4 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Ab7291, by Abcam (2 mentions)
Ab14711, by Abcam (2 mentions)
Ab14744, by Abcam (2 mentions)
Ab110246, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab33985, by Abcam (2 mentions)
Criterion tgx stain free gel, by Bio-Rad (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Ab151229, by Abcam (1 mentions)
A1978, by Merck Group (1 mentions)
P0260, by Agilent Technologies (1 mentions)
P0399, by Agilent Technologies (1 mentions)
Ab24733, by Abcam (1 mentions)
33 protocols using ab14714
1
Immunoblotting Analysis of Differentially Abundant Proteins
2
Western Blot Analysis of Skeletal Muscle
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Skeletal muscle and fibroblast homogenates were obtained according to previously described methodologies.30 (link) 30–40 μg (S1–S3) and 20 μg (S4) of whole-cell protein extracts were separated by SDS polyacrylamide (12%) electrophoresis and then wet transferred to polyvinyl difluoride (PVDF) membranes. For S4, a 4%–12% gradient gel was used. Immunological detection of proteins was carried out with the following primary antibodies: C1QBP (ab24733, Abcam), β-actin (A1978, Sigma), α-tubulin (ab7291, Abcam), and OXPHOS complex-specific antibodies (NDUFS3 [ab14711, Abcam], NDUFB8 [ab110242, Abcam], NDUFA9 [MS111, Molecular Probes], SDHA [459200, MitoSciences], SDHB [ab14714, Abcam], UQCRC2 [ab14745, Abcam], COXI [ab14705, Abcam], COXII [ab110258, Abcam], COXIV [ab14744, Abcam], and ATP5A [ab14748, Abcam]). Species-appropriate horseradish-peroxidase-conjugated secondary antibodies (DAKO, P0399, and P0260) were used.
3
Immunohistochemical Profiling of Tumor Microenvironment
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All immunohistochemistry was conducted on 5-µm sections of tumor and normal tissue. Morphological analysis was assessed using H&E, before analysis of any tumor immunosuppression activity, using the 22C3 pharmDx Kit (Agilent) against programmed cell death ligand-1 (PD-L1), according to manufacturer's instructions. T-cell infiltration was assessed with anti-CD3 (Roche 790-43141) and CD8 (Agilent M7103) antibodies according to the manufacturer's instructions, as was qualitative assessment of mitochondrial staining using a mouse monoclonal antibody against SDHB (Abcam ab14714).
4
Western Blotting Analysis of Mitochondrial Complexes
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Total proteins were extracted using radioimmunoprecipitation assay buffer. Protein concentration was assessed with a Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The same amount of protein (μg) was loaded in each lane. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride or nitrocellulose membrane and subsequently subjected to immunoblotting analysis using appropriate antibodies. The amount of loading was further determined using a western blotting housekeeping protein (lamin A, 1:5000 dilution). Using a horseradish peroxidase-conjugated secondary antibody, protein bands on the blots were visualized with enhanced chemiluminescent western blot detection reagent. Antibodies including anti-tubulin (Abcam, ab7291), anti-LDHB (Abcam, ab75167), anti-complex I (Abcam, ab110242), anti-complex II (Abcam, ab14714), anti-complex III (Abcam, ab14745), anti-complex IV (Abcam, ab14705), anti-complex V (Abcam, ab14748) were purchased from commercial company (Abcam, Cambridge, UK).
5
Protein Isolation and Western Blotting
6
Immunohistochemical Analysis of Mitochondrial Complexes
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Cryosections, 8‐μm thick, were cut and mounted on Superfrost Plus slides (J1800AMNZ, Thermo Scientific). Standard techniques were applied for enzyme histochemical analyses of COX, SDH and combined COX‐SDH [16 ]. For immunohistochemical studies of the mitochondrial respiratory chain complex, cryosections were fixed in 4% formaldehyde at 4°C for 10 min, washed in Tris‐buffered saline‐Tween 20 (TBS‐T) for 10 min, permeabilised in a graded methanol series (70% 10 min, 95% 10 min, 100% 20 min, 95% 10 min and 70% 10 min) and washed in TBS‐T for 5 min. For the study of MHC‐class I (HLA‐ABC), the cryosections were fixed with acetone. The tissue sections were processed in a Dako Autostainer using the EnVision FLEX DAB+ Substrate Chromogen System kit and incubated with the following primary antibodies for 1 h: anti‐NDUFB8 (complex I, ab110242, Abcam, UK, 1:100), anti‐SDHB (complex II, ab14714, Abcam, 1:500), anti‐MTCO1 (complex IV, ab14705, Abcam, 1:2000), anti‐VDAC1 (porin, a mitochondrial marker, ab14734, Abcam, 1:2000) and anti‐human HLA‐ABC (M0736, clone W6/32, Dako, 1:1000). These antibodies to subunits of complex I, II and IV of the respiratory chain have been well characterised and demonstrated to be reliable tools in studies on mitochondrial myopathies [17 (link), 18 (link), 19 (link)].
7
Immunohistochemical Analysis of Hypoxia Markers
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The staining procedure was performed using a Leica Bond III Stainer (Leica,). Slides were subjected exposed to 10 mM sodium citrate buffer, pH 6.0 for 20 min at 37°C. Slides were incubated with the appropriate primary antibody for 15 min, followed by Polymer Refine Detection System (Leica) processing, including hydrogen peroxidase block, secondary antibody polymer, 3,3’ diaminobenzidine and hematoxylin stain. Specimens were then rinsed for 5 min in tap water. Slides were dehydrated in increasing concentrations of ethyl alcohol and xylene prior to permanent coverslipping in xylene-based media. Primary antibodies were as follows: mouse anti-Hif1α (1:400, Novus Biological), mouse anti-HIF2α (Novus Biologicals, NB100-132, rabbit polyclonal, 1:700) mouse anti-H3K9me2 (1:750, Abcam), rabbit anti-H3K27me2 (ab24684, Abcam, 1:500), rabbit anti-5hmC (39769, Active Motif, 1:4000), and mouse anti-SDHB (ab14714, Abcam, 1:1000).
8
Immunoblotting Analysis of AKT and SDHB
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The extracted protein after boiled for 6 min was separated by 10% SDS-PAGE (BioRad) and then transferred onto a PVDF membrane (Millipore, USA). Then the PVDF membrane is blocked with 5% fat-free milk for 60 min. Primary antibodies were incubated at 4 °C overnight. In the following day, the TBST is used to wash out the primary antibodies and the membranes are incubated with secondary antibodies. The primary antibodies used in the experiment were as follows: rabbit anti-human AKT (1:1000, Cell Signaling Technology #4691S), rabbit anti-human phospho-AKT (Ser473) (1:1000, Cell Signaling Technology #4060S), mouse anti-β-actin (1:2000, Santa Cruz #sc-47778) and mouse anti-SDHB antibody (1:1500, Abcam #ab14714).
9
Mitochondrial Protein Immunoprecipitation
10
Immunohistochemical Analysis of Mitochondrial and Mismatch Repair Proteins
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Immunohistochemistry was performed as per usual clinical staining protocols on formalin-fixed, paraffin-embedded tissue sections cut at a thickness of 4 μm. Following deparaffinization and rehydration, antigen retrieval was performed with citrate buffer in a pressure cooker. Tissues were incubated with primary antibodies followed by secondary detection with commercial kits. The primary antibodies used were as follows: SDHA (1:800, clone 2E3; ab14715, Abcam), SDHB (1:300, clone 21A11; ab14714, Abcam), MLH1 (1:300, clone G1680728; MLH1-L-CE, Leica), PMS2 (1:300, clone MRQ-28; 288m-16, Cell Marque), MSH2 (1:400, clone FE11; NA27, Oncogene), and MSH6 (1:800, clone PU29; NCL-L-MSH6, Leica). The DAKO Envision plus detection system was used for SDHA and SDHB, whereas the Leica Novolink detection system was used for MLH1, PMS2, MSH2, and MSH6.
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