Mouse monoclonal anti gapdh sc 32233

Total proteins were extracted from different groups of U251 gliomaspheres with the same treatment as described in the DIGE analysis, and protein concentrations were quantified by BCA kit. Western blot procedures were carried out as we previously described [40 (link)], with minor modifications. Namely, after boiling for 5 min with loading buffer, the same amount of proteins from each groups were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with mouse monoclonal anti-ECE-1 antibody (sc-376017, Santa Cruz, CA, USA), mouse monoclonal anti-alpha-enolase (sc-101513, Santa Cruz, CA, USA), mouse monoclonal anti-cathepsin A (sc-73766, Santa Cruz, CA, USA) and mouse monoclonal anti-GAPDH (sc-32233, Santa Cruz, CA, USA) at 1:500 dilution. The immunoblots were developed by incubation with goat anti-mouse IgG-HRP (sc-2005, Santa Cruz, CA, USA) as the secondary antibody followed by ECL detection (GE Healthcare).

Total proteins were extracted from cells in different treatment groups and the concentrations were measured by the BCA kit. Protein samples (30 μg/lane) were separated by SDS-PAGE and transferred to PVDF membranes. The membrane was then incubated overnight at 4°C with different primary antibodies in blocking solution: mouse monoclonal anti-PADPR 10H (abcam, Cambridge, UK); mouse monoclonal anti-PARG antibody (sc-398563, Santa Cruz); mouse monoclonal anti-GAPDH (sc-32233, Santa Cruz). The immunoblots were then incubated with goat anti-mouse IgG-HRP (sc-2005, Santa Cruz) as secondary antibody followed by ECL detection (GE Healthcare).

The CFTR modulators ivacaftor and tezacaftor were from TargetMol (catalog ID: T2588 and T2263, respectively; Wellesley Hills, MA, USA). Elexacaftor was purchased from MedChemExpress (catalog ID: HY-111772; Monmouth Junction, NJ, USA). The final working concentration used for the CFTR modulators were as follows: Elexacaftor, 3 µM; tezacaftor, 10 µM; ivacaftor, 1 µM (when applied acutely during short-circuit current measurements or for the YFP assay) or 5 µM (for 24 h treatment).
The following antibodies were used: mouse monoclonal anti-CFTR (ab769, J.R. Riordan, University of North Carolina at Chapel Hill, and Cystic Fibrosis Foundation Therapeutics); mouse monoclonal anti-GAPDH (sc-32233; Santa Cruz Biotechnology, Inc.; RRID: AB_627679, Dallas, TX, USA); horseradish peroxidase (HRP)-conjugated anti-mouse IgG (ab97023; Abcam; RRID: AB_10679675, Cambridge, UK) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (0031460; ThermoFisher Scientific, Waltham, MA, USA). Vectors encoding WT-, S737F-, and F508del-CFTR variants were purchased from VectorBuilder (vector IDs available upon request; Neu-Isenburg, Germany).