This is a knockout-validated antibody summary, based on the publication "Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9", as cited below [1]. Other articles also indicates knockout validation of this antibody: "NCOA4 maintains murine erythropoiesis via cell autonomous and non-autonomous mechanisms" (Western blot, Figure S1B) [2] and "Thyroid hormone receptor beta and NCOA4 regulate terminal erythrocyte differentiation" (Western blot, figure 5a) [3]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Mouse monoclonal IgG1 (kappa light chain)

Company: Santa Cruz

Antibody: ARA70

Catalog number: sc-373739

Summary: Mouse monoclonal IgG1 (kappa light chain) against amino acids 1-300 mapping at the N-terminus of human ARA70 (NCOA4). Recommended for detection of ARA70 of human origin by western blot, immunoprecipitation, immunofluorescence and ELISA.

Western blot

Control and NCOA4 knockout H4 cell lines. Cells were lysed in RIPA buffer (#9806, Cell Signaling Technology) supplemented with sodium dodecyl sulfate (SDS, Boston BioProducts) to 1% final concentration, and protease inhibitor tablets (Complete EDTA-free, Roche).

5% non-fat dry milk (Bio-Rad) in TBS with 0.2% Tween-20 (Boston BioProducts).

1:1,000 dilution in 5% bovine serum albumin (BSA, Cell Signaling Technology) in TBS with 0.2% Tween-20 at 4°C overnight.

1:10,000 dilution HRP-conjugated secondary antibodies diluted in blocking solution at room temperature for 1 h.

Pico or West Femto SuperSignal ECL reagents (Pierce) and film (GE Healthcare).

Immunoblot analysis of control or clonal NCOA4 knockout H4 cell lines blotted with the indicated antibodies. Please see Figure 3j in the article [1].