According to the manufacturer's instructions, paraffin-embedded tissue samples from consenting patients were incubated overnight using the following primary antibodies (1:50-100): SHMT2 (12762, Cell Signaling Technology), β-catenin (D10A8) XP (8480, Cell Signaling Technology). The IHC images were captured by a microscope (Olympus, USA). To get the relative expression (%) of the target protein, the measurement parameters collected by Image J included integral optical density (IOD), area and average optical density (AOD). The AOD represented the levels of specific protein per unit area.
Shmt2
Manufactured by Cell Signaling Technology
Sourced in United States
SHMT2 is a laboratory product offered by Cell Signaling Technology. It is an enzyme involved in the folate-dependent one-carbon metabolism pathway. SHMT2 catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate.
Automatically generated - may contain errors
Lab products found in correlation
Shmt1, by Cell Signaling Technology (6 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Phenylmethylsulphonyl fluoride, by Merck Group (2 mentions)
Mthfd1, by Proteintech (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Tris glycine sds gels, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 bead, by Merck Group (2 mentions)
Phgdh, by Cell Signaling Technology (2 mentions)
Cybrfast 1 step rt qpcr lo rox kit, by Cytek Biosciences (2 mentions)
Phgdh, by Abcam (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Vinculin, by Cell Signaling Technology (2 mentions)
Cell signaling lysis buffer, by Cell Signaling Technology (2 mentions)
Novex 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
17 protocols using shmt2
1
Quantitative Immunohistochemical Analysis
2
Western Blot Analysis of One-Carbon Metabolism Enzymes
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from colon cancer cells and tissues with RIPA lysis buffer (Auragene Bioscience, China) supplemented with a protease inhibitor cocktail (Auragene Bioscience, China) and phenylmethanesulfonyl fluoride (PMSF; Auragene Bioscience, China). Equal amounts (10 μg) of proteins were subjected to SDS-PAGE, and then they were transferred to a PVDF Immobilon-P membrane (Millipore, USA). The membrane was blocked with 3% BSA-TBST at room temperature for 90 min. Additionally, the membrane was continuously probed with primary antibodies at 4 °C overnight. Next, the membranes were washed and incubated with specific secondary antibodies. A GAPDH antibody was used as a control, and SHMT2 (1:1000; Cell Signaling) antibodies were used for each group.
4
Immunoprecipitation and Analysis of Protein Complexes
5
Immunoprecipitation and Analysis of Protein Complexes
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All immunoprecipitations were performed as previously described 46 (link). Briefly, BRCC36 and Abraxas2 expressing cells were lysed in NETN 150 (0.5% NP40, 25 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA with 1mM PMSF). Flag-IPs were performed on lysate supernatants for 4 hours with agarose conjugated anti-Flag M2 beads (Sigma) prior to elution in 0.1 mM glycine (pH 2.5). Immunoblots were performed with the following rabbit polyclonal antibodies at 1:1000 dilution that were generated to recombinant proteins: MERIT40, Abraxas2, BRCC36 and BRCC45. Commercially available antibodies to HA epitope (HA.11 Covance) and SHMT2 (Cell Signaling) were used according to the manufacturer’s directions. An Abraxas2 deletion mutant (DM1) was used as control as described previously 12 .
For assessment of IFNAR1 ubiquitylation in cells, cells were lysed in buffer containing 2% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 50 mM sodium fluoride, 10mM N-Ethylmaleimide and protease inhibitors. After boiling for 10 mins, cell lysates were further diluted in immunoprecipiation buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton). IFNAR1 were immunoprecipitated with a corresponding antibody (Bethyl Laboratories A304-290A). Resulting samples were separated by SDS-PAGE and analyzed by immunoblotting using the anti-ubiquitin antibody (Lifesensor, VU101).
For assessment of IFNAR1 ubiquitylation in cells, cells were lysed in buffer containing 2% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 50 mM sodium fluoride, 10mM N-Ethylmaleimide and protease inhibitors. After boiling for 10 mins, cell lysates were further diluted in immunoprecipiation buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton). IFNAR1 were immunoprecipitated with a corresponding antibody (Bethyl Laboratories A304-290A). Resulting samples were separated by SDS-PAGE and analyzed by immunoblotting using the anti-ubiquitin antibody (Lifesensor, VU101).
6
Quantifying Oxidative Stress Markers
7
Whole-Cell Extract Protein Analysis
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To prepare whole-cell extracts, the cells were lysed using Pro-Prep buffer (Intron Biotechnology, Seoul, South Korea) containing phosphatase and protease inhibitors. Equal amounts of lysate were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk, the membranes were incubated with the indicated primary antibodies, washed, and incubated with the appropriate secondary antibodies. The following antibodies were used: SHMT2 (Cell Signaling Technology, Danvers, MA, no. 12762), IGFBP5 (Santa Cruz Biotechnology, sc-515116), vimentin (Thermo Fisher Scientific, Waltham, MA, no. MA5-11883), E-cadherin (24E10) (Cell Signaling Technology, no. 3195), and β-actin (Cell Signaling Technology, no. 3700). β-actin was used as the loading control for western blotting. Protein bands were detected using an enhanced chemiluminescence reagent.
8
Antibodies for Cellular Signaling Pathways
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Antibodies obtained were directed against the following: EGFR (Cell Signaling, #2232), p-EGFR Tyr1068 (Cell Signaling, #2236); EGFR/EGFRvIII cocktail antibody (Novocastra); PARP (Cell Signaling, #9542), cleaved PARP (Cell Signaling #5625); PSAT1 (Abcam ab154055); SHMT1 (Cell Signaling, #80715); SHMT2 (Cell Signaling, #12762); MTHFD1 (Abcam ab70203); MTHFD2 (Abcam ab151447); MTHFD2 (Abcam ab56772); LC3A (Cell Signaling, #4599); LC3B (Cell Signaling, #3868); and β-actin (Ambion). Reagents used are Chloroquine (CQ, Sigma) and Nicotinamide Adenine Dinucleotide reduced form (NADH, Nacalai Tesque).
9
Western Blot Analysis of Protein Expression
10
Quantifying Mitochondrial and Metabolic Gene Expression
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