Human Tissue slices were deparaffinized and hydrated by a series of xylene and alcohol treatment, and the procedure was performed as previously described13 (link). The slices were incubated with rabbit polyclonal anti-MPC1 (1:250; Abcam, Cambridge, UK) and anti-P-STAT3(Y705) (1:100; Abcam, Cambridge, UK); anti-SOX2 (1:100; CST, Danvers, MA, USA); anti-MMP2 (1:100; CST, Danvers, MA, USA) antibodies at 4 °C overnight, followed by incubation with avidin–biotin–peroxidase (DAKO). MPC1 expression using the following system: 0, 0–5% positive cells; 1, < 25% positive cells; 2, 25–50% positive cells; 3, 50–75% positive cells; and 4, 75–100% positive cells. The staining intensity was scored as follows: 0, no positive staining; 1, weak staining; 2, moderate staining; 3, strong staining. The final scores were obtained by multiplying the extent scores by intensity scores and analysis using the statistical X-tile software with score of 4 as the cutoff value14 (link).
Anti mpc1
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-MPC1 is a protein antibody that recognizes the Mitochondrial Pyruvate Carrier 1 (MPC1) protein. MPC1 is a key component of the mitochondrial pyruvate carrier, which is responsible for transporting pyruvate into the mitochondria. This antibody can be used for the detection and analysis of MPC1 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin peroxidase, by Agilent Technologies (3 mentions)
Anti p stat3 y705, by Abcam (1 mentions)
Anti lamin a c, by Proteintech (1 mentions)
Acy 1215, by Selleck Chemicals (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti hdac6, by Proteintech (1 mentions)
Anti hif 1α, by Proteintech (1 mentions)
Anti β catenin, by Proteintech (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Anti oct4, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Beyotime (1 mentions)
Anti sox2, by Abcam (1 mentions)
Anti nanog, by Abcam (1 mentions)
Anti flag, by Beyotime (1 mentions)
Anti mmp 7, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Abcam (1 mentions)
6 protocols using anti mpc1
1
Immunohistochemical Analysis of MPC1, P-STAT3, SOX2, and MMP2
2
Comprehensive Western Blot Analysis
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Western blotting was performed as previously described15 . The primary antibodies were as follows: anti-MPC1(1:1000; Abcam, Cambridge, UK); anti-GAPDH (1:1000 CST, Danvers, MA, USA); anti-COX IV (1:1000 CST, Danvers, MA, USA); anti-β-actin (1:1000 CST, Danvers, MA, USA); anti-P-STAT3 (Y705)(1:1000; CST, Danvers, MA, USA); anti-P-STAT3 (S727) (1:1000; CST, Danvers, MA, USA); anti-STAT3 (1:1000; CST, Danvers, MA, USA); anti-OCT4 (1:1000 CST, Danvers, MA, USA); anti-MMP2 (1:1000 CST, Danvers, MA, USA); anti-MMP3 (1:1000 CST, Danvers, MA, USA); anti-MMP7 (1:1000 CST, Danvers, MA, USA); anti-SOX2 (1:1000; CST, Danvers, MA, USA); anti-NANOG (1:1000; CST, Danvers, MA, USA).
3
MTT Assay with HDAC Inhibitors
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3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tubacin (HY-13428) was purchased from Med Chem Express (Monmouth Junction, NJ). ACY-1215 was purchased from Selleck (shanghai, China). The following anti-body were used: anti-MPC1, anti-NR2F2 (Abcam, Cambridge, MA, USA); anti-HDAC6, anti-HIF1α, anti-β-Catenin, anti-LaminA/C, anti-beta actin (Proteintech, Chicago, IL, USA); anti-Acetyl-β-Catenin (Lys49) #9534, anti-acetyl-α-Tubulin (Lys40) #3971 (Cell Signaling Technology, Beverly, Massachusetts, USA).
4
Western Blot Analysis of Stem Cell Markers
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Cells and tissue samples were lysed in RIPA lysis buffer (Beyotime, Jiangsu, China) supplemented with protease inhibitors (phenylmethylsulfonyl fluoride [PMSF]; Thermo Fisher Scientific, Waltham, MA, USA) on ice. Lysates were centrifuged at 12,000 g for 15 min at 4°C before aliquots of 20 μg proteins were electrophoresed on 15% SDS-PAGE gels (Bio-Rad Laboratories Inc., Hercules, CA, USA), transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA), and incubated overnight with primary antibodies: anti-MPC1 (Abcam, 1:500), anti-FLAG (Beyotime, 1:1,000), anti-Sox-2 (Abcam, 1:500), anti-Oct-4 (Abcam, 1:400), anti-Nanog (Abcam, 1:300), and anti-β-actin (Beyotime, 1:1,000). The membranes were then incubated with appropriate HRP-conjugated secondary antibody for 2 h at room temperature. Chemiluminescence was detected using SuperSignal West Femto Maximum Sensitivity Substrate (enhanced chemiluminescence, Thermo Fisher Scientific). β-actin was used as a loading control. All of the experiments were performed in triplicate.
5
Immunohistochemical Analysis of MPC1 and HIF1α
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Human tissue sections were deparaffinized and hydrated by a series of xylene and alcohol treatment. The sections were incubated with rabbit polyclonal anti-MPC1 (Abcam, Cambridge, UK), and mouse polyclonal anti-HIF1α (Abcam) antibodies at 4 °C overnight, followed by incubation with avidin-biotin-peroxidase (DAKO). MPC1 and HIF1α were considered positive by cytoplasmic staining and the expression levels were semi-quantitatively analyzed using a composite score system by assessing both the percentage and intensity of stained tumor cells. The percentage of positive cells was calculated in high-power fields (HPF) as follows: [10] sections with <1% positive cells were rated as 0; 1-25% positive cells as 1; 26-50% positive cells as 2; 51-75% positive cells as 3, and 76-100% positive cells as 4. The staining intensity was rated as follows: 1 for weak intensity; 2 for moderate intensity; and 3 for high intensity. Points for staining intensity and the percentage of positive cells were multiplied. Tumor specimens were classified into three groups according to overall scoring: 0-1 as negative expression, 2-4 as weak expression, and 6-12 as high expression. Total scores were expressed as: 0-4 as low and 6-12 as high. All sections were evaluated independently by two pathologists without knowledge of the identity of patients and the clinical outcome.
6
Xenograft Tumor Growth Assay
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All animal experiments were approved by the Institutional Animal Care and Use Committee of the Southwest Hospital, Third Military Medical University (Army Medical University). A total of 1 × 10 5 MPC1overexpressing or control cells were suspended in 50 μl PBS and subcutaneously injected into 6-week-old female NOD-SCID mice (Laboratory Animal Center, Southwest Hospital, Third Military Medical University, China). The size of resultant tumors was measured every 7 days for a month using a Vernier caliper and tumor volume was calculated as: shortest diameter 2 × longest diameter/2. The animals were sacrificed 30 days after tumor cell implantation and subcutaneous xenograft tumors were analyzed by IHC. Tumor tissue sections deparaffinized and hydrated by a series of xylene and alcohol treatment were incubated with rabbit polyclonal anti-MPC1 (Abcam), rabbit polyclonal anti-MMP7 (Cell signaling), and mouse polyclonal anti-HIF1α (Abcam) antibodies at 4 °C overnight, followed by incubation with avidin-biotin-peroxidase (DAKO).
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