The cells were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris-HCl at pH 7.4, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate and 1% NP-40) mixed with a protease and phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phenylmethylsulfonyl fluoride (PMSF) (Biosharp, Hefei, China) for 15 min on ice. The proteins were subjected to western blotting according to standard protocols. Antibodies were as follows: SIRT2 (HPA011165,Sigma), SIRT2 (s8447, Sigma), cMYC (ab32072, Abcam, Cambridge, UK), p293-PDHA1 (ab92696, Abcam), GAPDH (ab128915, Abcam), cleaved-caspase3 (9664s, CST, Danvers, MA, USA), cleaved-PARP (5625s, CST), PDHA1 (ab110334, Abcam), PHGDH (14719-1-AP, Proteintech, Chicago, IL, USA), PSAT1 (10501-1-AP, Proteintech), PSPH (14513-1-AP, Proteintech), HRP-conjugated anti-rabbit (7074, CST), and anti-mouse antibodies (7076, CST).
Ab92696
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab92696 is a laboratory product offered by Abcam. It serves as a tool for scientific research, but a detailed description of its core function is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab128915, by Abcam (2 mentions)
Ab110334, by Abcam (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
S8447, by Merck Group (1 mentions)
Ab32072, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Ab88078, by Abcam (1 mentions)
Ab207450, by Abcam (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Ab92547, by Abcam (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Dapi d1306, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl western blotting reagent, by Merck Group (1 mentions)
Sirt3, by Merck Group (1 mentions)
C myc, by Abcam (1 mentions)
Lysis buffer, by Biosharp (1 mentions)
Ab115730, by Abcam (1 mentions)
12 protocols using ab92696
1
Western Blot Analysis of Cellular Proteins
2
Protein Expression Quantification in Muscle Biopsies
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Protein samples from muscle biopsies were fractionated on SDS–9% PAGE and blotted with anti-β-F1-ATPase (1:1000), anti-Hsp60 (1:1000), anti-GAPDH (1:1000) and anti-PKM2 (1:1000) from [11 (link)], anti-IF1 (1:500) from [12 (link)], anti-LDH-A (1:1000), anti-GPD1 (1:1000) from [13 (link)], anti-PYGM (Abcam, ab88078; 1:1000), anti-PKM1 (Abcam, ab6191-5; 1:1000), anti phospho-PKM2 (Tyr105) (Cell Signaling, #3827; 1:1000), anti-PDHE1α (Invitrogene, 459400; 1:500), anti-phospho-PDHE1α (Ser293) (Abcam, ab92696; 1:1000), anti-PDK1 (Abcam, ab207450; 1:1000) and anti-β-actin (Sigma-Aldrich, A1978; 1:1000). Peroxidase-conjugated anti-mouse IgGs (Nordic Immunology; 1:3000) were used as secondary antibodies. The blots were revealed using the ECL® reagent (Amersham Pharmacia Biotech). The intensity of the bands was quantified using a Kodak DC120 digital camera and the Kodak 1D Analysis Software.
3
Western Blot Protein Extraction and Analysis
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Cells for protein extraction were grown in T75 flasks, washed with cold PBS, scraped, pelleted and frozen in −80 °C, before they were lysed using RIPA lysis buffer. 20 µg protein was loaded into 6–12% SDS-PAGE gels from Bio-Rad Laboratories (Hercules, CA, USA). Blotting was performed by using the Bio Rad Trans-Blot Turbo system and blocking was performed for 2 h in 5% milk in TBS-T (0.1% (v/v) Tween). Primary antibodies used were PDK1 (ab207450, Abcam, Cambridge, UK), and p-PDH (ab92696, Abcam), E-Cadherin (CDH1, 14472S, cell signaling), vimentin (VIM, ab92547, Abcam), LDHA (3582, Cell signaling). The housekeeping protein a-tubulin or vinculin was used as loading control when total protein staining was not available. Primary antibodies were removed, and the membranes were washed three times for 5 min with TBS-T before incubating them with secondary antibody for 1 h under agitation. The membranes were washed three times with TBS-T before they were examined. Complete blots can be found in Supplementary Figure S4 .
4
Metabolic Regulation of HEK293T and HeLa Cells
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HEK293T cells (ATCC, CRL-11268) were cultured in Dulbecco’s modified eagle medium (DMEM) (Gibco, 12430-054) containing 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, MA, USA, 10099141) and HeLa cells (ATCC®, CCL-2TM) were cultured in minimum essential medium (MEM) (Gibco, 11095-080) containing 1% NEAA (Gibco, 11140-050) and 10% FBS (Gibco, 10099141). Cells were transfected with plasmids using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, MA, USA, Cat. No. 12566014). Wild type HKII, HKII mutant, wild type PDHA1, PDHA1 S293A, and PDHA1 S293E were cloned into pcDNA3.1-Flag, pcDNA3.1-Myc, and pcDNA3.1-HA vectors, respectively. A HKII-specific siRNA (5’-TCGCATCTGCTTGCCTACTTCTTCA-3’) was used to knock down HKII gene, and a non-silencing siRNA oligonucleotide was used as a negative control. 3-Bromopyruvate (3BP, 16490-10G) and 2-deoxy-D-glucose (2-DG, D8375-10 mg) were purchased from Sigma-Aldrich, DAPI (D1306) from Invitrogen, anti-HKII antibody (Arg66209) from Arigobio, anti-PDHA1 (ab67592) and anti-phosphorylated PDHA1 (phosphor S293) (ab92696) antibodies from Abcam, and PDH enzyme microplate assay kit (ab109902) from Abcam.
5
Cardiac Protein Expression Profiling
6
Western Blot Analysis of Metabolic Proteins
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The cells were lysed using lysis buffer (Biosharp) to obtain total protein. Next, loading buffer containing 5% 2‐mercaptoethanol was mixed with the lysate. The proteins were denatured at 100°C for 10 minutes before being separated on 8%‐12% sodium dodecyl sulfate‐polyacrylamide gels. After transfer to PVDF membranes (Millipore), the membranes were incubated for 2 hours at room temperature in TBST containing 5% skim milk. Afterward, membranes were incubated with the corresponding primary antibodies overnight at 4°C according to the manufacturer's instructions. After treatment with the appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies, the membranes were incubated with ECL western blotting reagents (Millipore). The following antibodies were used: SIRT3 (s4072; Sigma), cMYC (ab32072; Abcam), HIF1α (36169; CST, Danvers, MA, USA), PDK1 (5662; CST), p‐PDHA1 (ab92696; Abcam), GAPDH (ab128915; Abcam), Actin (A5441; Sigma), PDHA1 (ab110334; Abcam), GLUT1 (ab115730; Abcam), Anti‐mouse IgG (7076; CST), and Anti‐rabbit IgG (7074; CST).
7
Co-expression Analysis of miR-155 Targets
8
Immunostaining of Drosophila Tissues
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Intact guts were fixed at room temperature for 20 min in PBS, 3.7% formaldehyde. All subsequent incubations were done in PBS, 4% horse serum, 0.2% Triton X-100 at 4°C following standard protocols. To visualize the three-dimensional arrangement of the internal organs inside the male body cavity, intact abdomens were fixed at room temperature for 20 min in PBS, 3.7% formaldehyde prior dissection and cuticle removal.
The following primary antibodies were used: chicken anti-GFP (ab13970, Abcam) 1/10000, mouse anti-GFP (11814460001, Roche) 1/1000, chicken anti-beta Galactosidase (ab9361, Abcam) 1/200, rabbit anti-phospho-Histone H3 Ser10 (9701L, Cell Signaling Technology) 1/500, mouse anti-Fas3 (7G10, DSHB) 1/50, rabbit anti-Pyruvate dehydrogenase E1-alpha subunit (phospho S293) (ab92696, Abcam) 1/200, rabbit anti-Aconitase 2 (ab83528, Abcam) 1/200, rabbit anti-cleaved Drosophila Dcp-1 (Asp216) (9578S, Ozyme) 1/500, and rhodamine Phalloidin (R415, ThermoFisher scientific) 1/1000. Fluorescent secondary antibodies (FITC-, Cy3- and Cy5-conjugated) were obtained from Jackson Immunoresearch. Vectashield with DAPI (Vector Labs) was used to stain DNA.
The following primary antibodies were used: chicken anti-GFP (ab13970, Abcam) 1/10000, mouse anti-GFP (11814460001, Roche) 1/1000, chicken anti-beta Galactosidase (ab9361, Abcam) 1/200, rabbit anti-phospho-Histone H3 Ser10 (9701L, Cell Signaling Technology) 1/500, mouse anti-Fas3 (7G10, DSHB) 1/50, rabbit anti-Pyruvate dehydrogenase E1-alpha subunit (phospho S293) (ab92696, Abcam) 1/200, rabbit anti-Aconitase 2 (ab83528, Abcam) 1/200, rabbit anti-cleaved Drosophila Dcp-1 (Asp216) (9578S, Ozyme) 1/500, and rhodamine Phalloidin (R415, ThermoFisher scientific) 1/1000. Fluorescent secondary antibodies (FITC-, Cy3- and Cy5-conjugated) were obtained from Jackson Immunoresearch. Vectashield with DAPI (Vector Labs) was used to stain DNA.
9
Western Blot Analysis of Protein Expression
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Total protein (10 μg/mL) extracted 72 h post-transfection was separated by 4–15% Mini-Protean TGX Precast gel from Bio-Rad Laboratories at 80 V. Protein was transferred to PVDF membrane, then blocked with 5% milk and 1% BSA in buffer consisting of 150 mM NaCl, 20 mM Tris -HCl, pH 7.5 and 0.1% (v/w) Tween for 1 h. The blot was probed with PDHA1 (1:500; # ab92696, Abcam, UK), GCK (1:500; #ab37796, Abcam, UK), or Cyclophilin B (1:2000; # ab16045 Abcam, UK) antibodies, and incubated overnight at 4 °C. Horseradish peroxidase conjugated goat anti-rabbit IgG, HRP-linked antibody (1:10 000; #7074; Cell Signaling Technology, Danvers, MA, USA) was used to detect the primary antibodies. Super Signal West Pico Chemiluminescent Substrate or Super Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific, MA, USA) and AlphaImager (ProteinSimple, CA, USA) was used to detect protein and quantification was done by using FluorChem SP software (Protein Simple, CA, USA).
10
Mitochondrial Protein Analyses by Western Blot
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