Indicated cells were lysed with RIPA buffer. Protein lysates were electrophoresed through SDS polyacrylamide gel, followed by transferring to PVDF membranes (Millipore, USA). The membranes were blocked with non-fat dry milk at room temperature and then incubated with primary antibodies at 4°Covernight. The primary antibodies included: FGFR1, PDHK1, P-PDHK1, LDHA, P-LDHA, MMP-1 and β-actin (Cell Signaling Technology). Membranes were then washed and incubated with secondary antibodies. Proteins were visualized by the electrochemiluminescence (ECL) western blot substrate detection (Pierce).
Pdhk1
Manufactured by Cell Signaling Technology
PDHK1 is a protein kinase enzyme that plays a key role in regulating the activity of the pyruvate dehydrogenase complex. It is involved in the phosphorylation and inactivation of the pyruvate dehydrogenase complex, which is a crucial step in the conversion of pyruvate to acetyl-CoA, a key intermediate in cellular energy metabolism.
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Lab products found in correlation
Fgfr1, by Cell Signaling Technology (1 mentions)
P ldha, by Cell Signaling Technology (1 mentions)
Mmp 1, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Golph3, by Proteintech (1 mentions)
Mtor substrates antibody sampler kit, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Hif 1α, by Proteintech (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Hydroxy hif 1α pro564, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Anti ha tag, by Abcam (1 mentions)
Hif 2α, by Novus Biologicals (1 mentions)
7 protocols using pdhk1
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Quantification of Cell Signaling Proteins
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Western blot was carried out as described [31 (link)]. The primary antibodies were used at the following dilutions: STK25 (1:1000, Cat #25821–1-AP, Proteintech), GOLPH3 (1:1000, Cat #19112–1-AP, Proteintech), PDHK1 (1:1000, Cat #3820, Cell Signaling Technology), HK2 (1:1000, Cat #2867, Cell Signaling Technology), LDHA (1:1000, Cat #2012, Cell Signaling Technology), AKT (1:1000, Cat #9272, Cell Signaling Technology), p-AKT (1:1000, Cat #9271, Cell Signaling Technology), mTOR Substrates Antibody Sampler Kit (1:1000, Cat #9862, Cell Signaling Technology), p70S6K (1:1000, Cat #2708, Cell Signaling Technology), p-p70S6K (1:200, Cat #9234, Cell Signaling Technology), and GOLPH3 (1:1000, Cat #19112–1-AP, Proteintech). The protein levels were normalized to β-actin (1,3000, Cat #A1978, Sigma-Aldrich). The densities of the proteins were quantified with ImageJ software.
3
Protein Expression Analysis by Western Blot
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Cells were lysed with PRO-PREPTM Protein Extraction for 1 h. Lysates were collected after centrifugation at 12,000 × g for 10 min at 4 °C. Equal amounts of protein measured by bovine serum albumin (BSA) were boiled for 5 min, and fractionated by SDS-PAGE. Primary antibodies against the following proteins were used: MCAD (Proteintech); HIF-1α, PDHK-1, Caspase8, BID, Caspase9, Caspase3, PARP and p53 (Cell Signaling Technology; CST). HRP-conjugated anti-mouse and anti-rabbit (CST) secondary antibody were used. In all Western blotting experiments, the blots were re-probed with anti-β-actin antibody to ensure equal protein loading. Bands were visualized with the EZ-Western Lumi Pico reagents according to the manufacturer’s instructions.
4
Western Blotting of Hypoxia Signaling Proteins
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Cells were lysed in a buffer consisting of Tris–HCl pH 7.4 (50 mM), NaCl (150 mM), Triton X-100 (1%), EDTA (2 mM), β-glycerophosphate (40 mM), PMSF (1 mM), NaF (10 mM), sodium orthovanadate (250 μM), and a protease inhibitor cocktail (Roche). Hypoxic samples were lysed inside the hypoxia chamber to prevent sample reoxygenation. Lysates were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes that were blotted with primary antibodies. Blots were further incubated with secondary horseradish peroxidase-conjugated antibodies (Cell Signaling) and stained with enhanced chemiluminescence reagent (Amersham). Chemiluminescence was detected on either film or using a ChemiDoc XRS+ System (Bio-Rad) and quantified using either Image J (https://imagej.nih.gov/ij/download.html ) or the Bio-Rad Image Lab Software (https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z ). The primary antibodies used were as follows: β-actin (Abcam, #ab6276, 1:10,000 dilution), HIF-1α (BD Biosciences, #610958, 1:1000 dilution), Hydroxy-HIF-1α (Pro564) (Cell Signaling, #3434, 1:1000 dilution), anti-HA tag (Abcam #ab9110, 1:1000 dilution), HIF-2α (Novus Biological, #NB-100-122, 1:1000 dilution), and PDHK1 (Cell Signaling, #C47H1, 1:1000 dilution).
5
Evaluating Metabolic Reprogramming Mechanisms
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Diethylnitrosamine (DEN, 73861), CCl4 (1601168), Oligomycin (O4876), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, C2920), antimycin A (A8674), rotenone (R8875), 2-deoxy-d -glucose (2-DG, D8375), PNGase F (G5166) and Swainsonine (S9263) and D-(+)-Glucose (G7021) were obtained from Sigma (St. Louis, MO, USA). SYBR green was purchased from Bio-Rad (Hercules, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit, anti-mouse immunoglobulin G were obtained from Beyotime (Haimen, Jiangsu, China). Glucose deficient DMEM medium and Trizol Reagent were obtained from Life Technologies (Carlsbad, CA, USA). Tunicamycin (B7417) was obtained from ApexBio Technology (Houston, TX, USA). UNC-2250 (S7342) was purchased from Selleck Chemicals (Houston, TX, USA). The antibodies used in this study are: MerTK (abcam; ab52968), Phospho-MerTK (abcam; ab14921), Akt (Cell Signaling Technology; 4691), Phospho-Akt (Ser473) (Cell Signaling Technology; 4060), Phospho-GSK3β (Cell Signaling Technology; 5558S), PGK1 (ZN; 501965), LDHA (ZN; 501146), PFKM (ZN; 505477), PKM2 (ZN; 505477), PDHK1 (Cell Signaling Technology; 3820S), Lamin B (Proteintech; 12987-1-AP), GAPDH (Abmart; M20028) and β-Actin (Proteintech; 60008-1-1 g).
6
Mitochondrial Protein Expression Analysis
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Cells were lysed in RIPA buffer (50 mM Tris-HCI, 150 mM Nacl, 0.1% SDS, 1.5% NP40, 0.5% deoxycholate, 2 mM Mgcl2) containing proteinase inhibitor for 30 min at 4 °C, then boiled in the SDS-PAGE loading buffer (P0015, Beyotime). Equal amounts of protein from each sample were subject to 10% SDS-PAGE and immunoblot analysis. Antibody used in current study includes anti-pyruvate dehydrogenase (PDHA, #3205, Cell signaling technology, 1:1000), anti-fumarate hydratase (FH, #4567, Cell signaling technology, 1:1000), anti-citrate synthase (CS, #14309, Cell signaling technology, 1:1000), anti-pyruvate dehydrogenase kinase 1 (PDHK1, #3820, Cell signaling technology, 1:1000), anti-TOM20 (#11802-1-AP, Proteintech, 1:1000), and anti- Actin (#AC026, Abclonal, 1:1000).
7
DRG Protein Extraction and Western Blot Analysis
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Protein was extracted from the L4-6 DRGs in lysis buffer (50 mM Tris HCl, 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA at pH 7.4) containing protease and phosphatase inhibitor mixtures with an ultrasonicator on ice, and cleared of cellular debris by centrifugation at 14,000 relative centrifugal force for 15 min at 4°C. Fifteen micrograms of protein per well were loaded and separated by standard 7.5% or 10% SDS-PAGE. Proteins were transferred to Immobilon-P membranes (Millipore Sigma, Cat # IPVH00010) and then blocked with 5% dry milk for 3 h at room temperature. The blots were incubated with primary antibody overnight at 4°C and detected the following day with donkey anti-rabbit or goat anti-mouse antibody conjugated to horseradish peroxidase (1:10,000, Jackson Immunoresearch, Cat # 711–036-152, Cat # 115–036-062). Signal was detected by enhanced chemiluminescence on films. For assessment of phospho-proteins membranes were stripped and reprobed for total protein of interest for normalization. Densitometric analyses were done using UN-SCAN-IT 7.1 software (Silk Scientific Corp.). Primary antibodies include phospho-PDH Ser300 (1:1000, Millipore Sigma, Cat # ABS192), PDH, LDHA, PDHK1 (1:1000 Cell Signaling Technology, Cat # 3205, 3582, 3820), and beta-III-tubulin (1:50,000 Promega, Cat # G7121).
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