Protein concentrations in cell and tissue lysates were determined by Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA). Proteins were resolved using reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, then transferred to polyvinylidene difluoride (PVDF) membranes followed by incubation with the indicated primary antibody. PKM1 (#7067), PKM2 (#4053), p-STAT3 (#8119), STAT3 (#4904), IKKε (#3416) and histone H3 (#4499) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). β-actin antibody was acquired from Sigma-Aldrich. Subsequently, membranes were incubated with peroxidase-conjugated secondary antibodies and visualized using chemiluminescence (Pierce, Rockford, IL, USA). Non-reducing gel electrophoresis assays in the presence of the disuccinimidyl suberate (DSS) crosslinker (Thermo Scientific, MA, USA) were performed to evaluate the formation of tetrameric PKM2. Densitometric analyses were performed using Image J Software. Values were normalized to corresponding β-actin bands.
Disuccinimidyl suberate dss crosslinker
Manufactured by Thermo Fisher Scientific
Sourced in United States
Disuccinimidyl suberate (DSS) is a homobifunctional N-hydroxysuccinimide (NHS) ester crosslinker. It can be used to covalently link primary amine-containing molecules.
Automatically generated - may contain errors
Lab products found in correlation
Histone h3 4499, by Cell Signaling Technology (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Hp dna transfection reagent, by Roche (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Taqman universal pcr master mix, by Roche (1 mentions)
Protein a and g dynabeads, by Thermo Fisher Scientific (1 mentions)
Edem1, by Proteintech (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Calnexin, by Stressgen (1 mentions)
Calreticulin, by Stressgen (1 mentions)
2 protocols using disuccinimidyl suberate dss crosslinker
1
Quantifying Protein Levels and PKM2 Oligomerization
2
Antibody-Based Detection of ER Stress Proteins
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Rabbit polyclonal antibodies were used to detect ERdj3, EDEM1, and ERp57 (Proteintech, Chicago, IL); others were to detect AAT (DAKO, Carpentaria, CA), GAPDH (Santa Cruz, Dallas, TX), Lamp1, P62, LC3B (all from Cell Signaling, Danvers, MA), and calnexin and calreticulin (both from Stressgen biotechnologies, San Diego, CA). Bafilomycin A1, MG132, Brefeldin A, and mouse monoclonal anti‐β‐actin were purchased from Sigma (St. Louis, MO), and 2C1 against human AAT polymers was purchased from Hycult biotech (Netherlands). Mouse monoclonal antibodies against BiP and AAT were purchased from BD bioscience (San Jose, CA) and R&D systems (Minneapolis, MN), respectively. Alexa Fluor 488 goat anti‐mouse IgG and Alexa Fluor 594 goat anti‐rabbit IgG were purchased from Invitrogen. Primers, Dynabeads protein A and G, Superscript VILO cDNA synthesis kit, and ERdj3 siRNA were purchased from Life Technologies, and HP DNA transfection reagent and TaqMan Universal PCR Master Mix were purchased from Roche Applied Science. Disuccinimidyl suberate (DSS) cross‐linker was purchased from Thermo Fisher scientific (Waltham, MA).
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